Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with cancer vaccine

Abstract

Provided are methods for treating cancer in a patient in need thereof with TILs in combination with a cancer vaccine.

Description

Attorney Docket No.: 116983-5131-WO TREATMENT OF CANCER PATIENTS WITH TUMOR INFILTRATING LYMPHOCYTE THERAPIES IN COMBINATION WITH CANCER VACCINE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 551,473, filed on February 8, 2024, which are hereby incorporated by reference in their entireties. BACKGROUND OF THE INVENTION

[0002] Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol.2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol.2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol.2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother.2003, 26, 332-42. A number of approaches to improve responses to TIL therapy in melanoma and to expand TIL therapy to other tumor types have been explored with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol.2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol.2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res.2011, 17, 4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional methods of treatment are needed (Kverneland, et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Cancer vaccines have a long history, but despite some promising early stage data have disappointed. Similarly, clinical data from neoantigen ‘selected’ cell therapy programs has also underwhelmed. Advances in DNA and mRNA technology have made personalized cancer vaccines targeting multiple neoantigens more feasible. There are cancer vaccine combinations (including vaccines encoding up to 34 neoantigens) in the clinic. 1DB1 / 154817694.1Attorney Docket No.: 116983-5131-WO BRIEF SUMMARY OF THE INVENTION

[0004] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient and dividing the tumor sample into a first portion and a second portion; b) expanding a population of TILs from the first portion of the tumor sample into a therapeutic population of TILs; c) making a cancer vaccine using the second portion of the tumor sample; d) administering the therapeutic population of TILs to the patient; and e) administering the cancer vaccine to the patient.

[0005] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient; b) making a cancer vaccine using the tumor sample; c) administering the cancer vaccine to the patient; d) resecting a second tumor sample from the patient after the patient responds to the cancer vaccine for a period of time; e) expanding a population of TILs from the second tumor sample into a therapeutic population of TILs; and f) administering the therapeutic population of TILs to the patient.

[0006] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient; b) making a first cancer vaccine using the tumor sample; c) administering the first cancer vaccine to the patient; d) resecting a second tumor sample from the patient after the patient responds to the cancer vaccine for a period of time and dividing the second tumor sample into a first portion and a second portion; e) expanding a population of TILs from the first portion of the second tumor sample into a therapeutic population of TILs; f) making a second cancer vaccine using the second portion of the second tumor sample; g) administering the therapeutic population of TILs to the patient; and h) administering the second cancer vaccine to the patient.

[0007] In some embodiments, making a cancer vaccine comprises: i) identifying between 1- 1,000 personalized cancer neoantigens by analyzing the second portion of the tumor sample; and ii) preparing a cancer vaccine wherein the cancer vaccine comprises portions of the 1- 1,000 personalized cancer neoantigens. In some embodiments, analyzing the second portion of the tumor sample comprises obtaining the transcriptome of a tumor cell in the second portion of the tumor sample. In some embodiments, analyzing the second portion of the DB1 / 154817694.1 2Attorney Docket No.: 116983-5131-WO tumor sample further comprises comparing the transcriptome of the tumor cell to the transcriptome of a reference cell. In some embodiments, the reference cell is a non-tumor cell in the second portion of the tumor sample. In some embodiments, obtaining the transcriptome of the tumor cell comprises single-cell sequencing. In some embodiments, the cancer vaccine comprises portions of 1-200 personalized cancer neoantigens. In some embodiments, the cancer vaccine comprises portions of 1-100 personalized cancer neoantigens.

[0008] In some embodiments, the cancer vaccine is a nucleic acid cancer vaccine comprising one or more nucleic acids each having one or more open reading frames. In some embodiments, the cancer vaccine comprises one or more nucleic acids each having one or more open reading frames encoding 1-1,000 peptide epitopes, wherein each of the peptide epitopes is portion of one of the 1-1,000 personalized cancer neoantigens from the patient. In some embodiments, the minimum length of any peptide epitope is 8-13 amino acids. In some embodiments, the maximum length of any peptide epitope is 31-35 amino acids. In some embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 1-1,000 peptide epitopes.

[0009] In some embodiments, the cancer vaccine is a DNA cancer vaccine. In some embodiments, the cancer vaccine is an RNA cancer vaccine. In some embodiments, the cancer vaccine is an mRNA cancer vaccine, and wherein the one or more nucleic acids are mRNA. In some embodiments, the one or more mRNA each comprise a 5' UTR and / or a 3' UTR. In some embodiments, the one or more mRNA each comprise a poly-A tail. In some embodiments, the poly-A tail comprises about 100 nucleotides. In some embodiments, the one or more mRNA each comprise a cap structure or a modified cap structure. In some embodiments, the cap structure or the modified cap structure is a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, or a 5' cap-2 structure. In some embodiments, the one or more mRNA comprise at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1- methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 5- DB1 / 154817694.1 3Attorney Docket No.: 116983-5131-WO methylpseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, and 2'-O-methyl uridine. In some embodiments, the one or more mRNA is fully modified.

[0010] In some embodiments, the one or more nucleic acids encode 1-5 peptide epitopes, 5- 10 peptide epitopes, 10-20 peptide epitopes, 20- 30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-200 peptide epitopes, 200-300 peptide epitopes, 300-400 peptide epitopes, 400-500 peptide epitopes, 500-600 peptide epitopes, 600-700 peptide epitopes, 700-800 peptide epitopes, 800-900 peptide epitopes, or 900-1,000 peptide epitopes. In some embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 5-130 peptide epitopes.

[0011] In some embodiments, one or more of the following conditions are met: a) the 1- 1,000 peptide epitopes are interspersed by cleavage sensitive sites; and / or b) each peptide epitope is linked directly to one another without a linker; and / or c) each peptide epitope is linked to one another with a single amino acid linker; and / or d) each peptide epitope is linked to one another with a short peptide linker; and / or e) each peptide epitope comprises 8-35 amino acids and includes one or more SNP mutations; and / or f) each peptide epitope comprises 8-35 amino acids and includes a mutation causing a unique expressed peptide sequence; and / or b) none of the peptide epitopes have a highest affinity for class II MHC molecules from a subject; and / or c) the nucleic acid encoding the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes; and / or d) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; and / or e) no class II MHC molecule peptide epitopes are present; and / or f) at least 30% of the peptide epitopes have a highest affinity for class I MHC molecules and / or class II MHC class molecules from a subject; and / or g) at least 50% of the peptide epitopes have a probability percent rank greater than 0.5% for HLA-A, HLA-B, and / or DRB1. In some embodiments, at least one of the peptide epitopes is a predicted T cell reactive epitope. In some embodiments, at least one of the peptide epitopes is a predicted B cell reactive epitope. In some embodiments, the peptide epitopes comprise a combination of predicted T cell reactive epitopes and predicted B cell reactive epitopes. In some embodiments, the peptide epitopes are predicted T cell reactive epitopes and / or predicted B cell reactive epitopes. In some embodiments, at least one of the peptide epitopes is a DB1 / 154817694.1 4Attorney Docket No.: 116983-5131-WO predicted neoepitope. In some embodiments, at least one nucleic acid has an open reading frame encoding at least a fragment of one or more traditional cancer antigens or one or more cancer / testis antigens. In some embodiments, each nucleic acid is formulated in a lipid nanoparticle. In some embodiments, each nucleic acid is formulated in a different lipid nanoparticle. In some embodiments, each nucleic acid is formulated in the same lipid nanoparticle. In some embodiments, the total length of the one or more nucleic acids encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700- 800 amino acids, 800-900 amino acids, 900-1,000 amino acids, 1,000-2,000 amino acids, 2,000-3,000 amino acids, 3,000-4,000 amino acids, 4,000-5,000 amino acids, 5,000-6,000 amino acids, 6,000-7,000 amino acids, 7,000-8,000 amino acids, 8,000-9,000 amino acids, or 9,000-10,000 amino acids. In some embodiments, the cancer vaccine is a peptide cancer vaccine.

[0012] In some embodiments, the cancer vaccine is administered at a dosage level sufficient to deliver between 0.02-1.0 mg of the cancer vaccine to the subject. In some embodiments, the cancer vaccine is administered to the patient after the administration of the therapeutic population of TILs. In some embodiments, the cancer vaccine is administered to the patient one week to twelve weeks after the administration of the therapeutic population of TILs. In some embodiments, the cancer vaccine is administered to the patient two weeks after the administration of the therapeutic population of TILs. In some embodiments, the cancer vaccine is administered to the patient once, twice, three times, four times, five times, six times, seven times, eight times, ten times, twelve times, or more. In some embodiments, the cancer vaccine is administered once weekly (QW), once every 2 weeks (Q2W), every 3 weeks (Q3W), or every 4 weeks (Q4W). In some embodiments, the cancer vaccine is administered Q3W for nine times. In some embodiments, the cancer vaccine is administered by intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous administration. In some embodiments, the cancer vaccine is administered by intramuscular administration. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, metastatic melanoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), a solid malignancy that is micro satellite high (MSI H) / mismatch repair (MMR) deficient, renal cancer, gastric cancer, and tumor mutational burden high tumors. In some DB1 / 154817694.1 5Attorney Docket No.: 116983-5131-WO embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), metastatic melanoma, and head and neck squamous cell carcinoma (HNSCC).

[0013] In some embodiments, the method provides for the manufacture of a medicament for use in the treatment of cancer BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1: Exemplary Gen 2 (process 2A) chart providing an overview of Steps A through F.

[0015] Figure 2A-2C: Process flow chart of an embodiment of Gen 2 (process 2A) for TIL manufacturing. DETAILED DESCRIPTION OF THE INVENTION

[0001] Described herein is a combination of tumor infiltrating lymphocyte (TIL) therapy and cancer vaccine made of neoantigens, wherein the TIL product and the cancer vaccine are produced from the same tumor sample. In some embodiments, the subject TIL + vaccine combination boosts an immune response after TIL infusion. In some emodiments, the TIL + cancer vaccine combo further enhances TIL persistence and activity in the neoantigen- reactive subset without adding extra delays to TIL cell therapy treatment. In exemplary embodiments, the vaccine enhances (‘boost’) the neoantigen-targeted TIL, but not other TIL that may be reacting against non-mutated tumor associated antigens.

[0002] Therefore, in some embodiments, provided herein is a method for treating a cancer patient, the method comprising: a) resecting a tumor sample from the patient and dividing the tumor sample into a first portion and a second portion; b) expanding a population of TILs from the first portion of the tumor sample into a therapeutic population of TILs; c) making a cancer vaccine using the second portion of the tumor sample; d) administering the therapeutic population of TILs to the patient; and e) administering the cancer vaccine to the patient. DB1 / 154817694.1 6Attorney Docket No.: 116983-5131-WO

[0003] In some embodiments, the manufacturing process is divided into two parallel paths at the time of tumor resection: a portion of the tumor ise used to make Gen 2 TIL for infusion (see Section III on Gen 2 TIL Manufacturing Processes); and the remaining tumor is sent for whole genome sequencing to identify potential neoantigens, and then to create a DNA or RNA vaccine for these neoantigens (see Section II on Cancer Vaccine Manufacturing Processes). I. Definitions

[0004] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0005] The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, a plurality of TILs) to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0006] The term “in vivo” refers to an event that takes place in a subject's body.

[0007] The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0008] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of surgery or treatment. DB1 / 154817694.1 7Attorney Docket No.: 116983-5131-WO

[0009] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs.

[0010] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 106to 1 X 1010in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 × 108cells. REP expansion is generally done to provide populations of 1.5 × 109to 1.5 × 1010cells for infusion.

[0011] By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[0012] By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

[0013] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. DB1 / 154817694.1 8Attorney Docket No.: 116983-5131-WO

[0014] The term “cryopreservation media” or “cryopreservation medium” refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof. The term “CS10” refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name “CryoStor® CS10”. The CS10 medium is a serum-free, animal component-free medium which comprises DMSO. In some embodiments, the CS10 medium comprises 10% DMSO.

[0015] The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to, closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient.

[0016] The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[0017] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti- CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0018] The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID DB1 / 154817694.1 9Attorney Docket No.: 116983-5131-WO NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No.86022706. TABLE 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody). Identifier Sequence (One-Letter Amino Acid Symbols)known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol.2004, 172, 3983-88 and Malek, Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL- 2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H- fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which may be prepared by methods known in the art, such as the DB1 / 154817694.1 10Attorney Docket No.: 116983-5131-WO methods described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Patent Nos.4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0001] In some embodiments, an IL-2 form suitable for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the invention are described in U.S. Patent Application Publication Nos. US 2020 / 0181220 A1 and US 2020 / 0330601 A1, the disclosures of which are incorporated by reference herein. In some embodiments, and IL-2 form suitable for use in the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, DB1 / 154817694.1 11Attorney Docket No.: 116983-5131-WO F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L- lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO- lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2- amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In some embodiments, the IL-2 conjugate has a decreased affinity to IL-2 receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Rα relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 1-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300- fold, 500-fold, 1000-fold, or more relative to a wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the additional conjugating moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof. In some embodiments, each of the water- soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan DB1 / 154817694.1 12Attorney Docket No.: 116983-5131-WO sulfate (HS), dextrin, or hydroxyethyl-starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises polyamine. In some embodiments, the conjugating moiety comprises a protein. In some embodiments, the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′- dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′- dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g.1,5- difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′- dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene- bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3-(2- pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate DB1 / 154817694.1 13Attorney Docket No.: 116983-5131-WO (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo- LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl- 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ- maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4- iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N- hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4- azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4- azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o- nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3′- dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo- sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7- azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4- (iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4- (ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal (APG). In some DB1 / 154817694.1 14Attorney Docket No.: 116983-5131-WO embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the invention is pegylated as disclosed in U.S. Patent Application Publication No. US 2020 / 0181220 A1 and U.S. Patent Application Publication No. US 2020 / 0330601 A1. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety DB1 / 154817694.1 15Attorney Docket No.: 116983-5131-WO comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.

[0002] In some embodiments, an IL-2 form suitable for use in the invention is nemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes, Inc. Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor α-chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser]-mutant (1-59), fused via a G2peptide linker (60- 61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG3S peptide linker (133-138) to human interleukin 2 receptor α-chain (IL2R subunit alpha, IL2Rα, IL2RA) (1- 165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, glycoform alfa. The amino acid sequence of nemvaleukin alfa is given in SEQ ID NO:6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168- 199 or 168-197 (using the numbering in SEQ ID NO:6), and glycosylation sites at positions: N187, N206, T212 using the numbering in SEQ ID NO:6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the invention, is described in U.S. Patent Application Publication No. US 2021 / 0038684 A1 and U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. In some embodiments, an IL-2 form suitable for use in the invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the invention has the amino acid sequence given in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some DB1 / 154817694.1 16Attorney Docket No.: 116983-5131-WO embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. Optionally, in some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having the receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, wherein the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 and wherein the half-life of the fusion protein is improved as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. TABLE 2. Amino acid sequences of interleukins. Identifier Sequence (One-Letter Amino Acid Symbols)DB1 / 154817694.1 17Attorney Docket No.: 116983-5131-WO SEQ ID NO:9 MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH 60 recombinant EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120 human IL-4 MREKYSKCSS 130 (rhIL-4)antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosures of which are incorporated by reference herein. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of: a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain DB1 / 154817694.1 18Attorney Docket No.: 116983-5131-WO comprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:29; and a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:38.

[0021] In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR1 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR3 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR3 of the VL, wherein the IL-2 molecule is a mutein.

[0022] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of a CDR sequence. The replacement by the IL-2 molecule can be the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region the CDR. A replacement by the IL-2 molecule can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequences.

[0023] In some embodiments, an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, an IL-2 molecule is engrafted indirectly into a CDR with a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0024] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein comprising an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent DB1 / 154817694.1 19Attorney Docket No.: 116983-5131-WO Application Publication No. US 2020 / 0270334 A1, the disclosure of which is incorporated by reference herein.

[0025] In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:25. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:16. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine engrafted protein comprises a VHregion comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein comprises a VLregion comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a VHregion comprising the amino acid sequence of SEQ ID NO:28 and a VL region comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. DB1 / 154817694.1 20Attorney Docket No.: 116983-5131-WO 2020 / 0270334 A1, or variants, derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine engrafted protein described herein has a longer serum half-life that a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule. In some embodiments, the antibody cytokine engrafted protein described herein has a sequence as set forth in Table 3. TABLE 3: Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO:13 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML IL-2 60DB1 / 154817694.1 21Attorney Docket No.: 116983-5131-WO SEQ ID NO:28 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY V 60 KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSS 253 SEQ ID NO:29 QMILNGINNY KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR Heavy chain 60 PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG 120 WIRQPPGKAL EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC 180 ARSMITNWYF DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 240 TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR 300 VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK 360 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK 420 TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533 SEQ ID NO:30 KAQLSVGYMH 10 LCDR1 kabat SEQ ID NO:31 DTSKLAS 7 LCDR2 kabat SEQ ID NO:32 FQGSGYPFT 9 LCDR3 kabat SEQ ID NO:33 QLSVGY 6 LCDR1 chothia SEQ ID NO:34 DTS 3 LCDR2 chothia SEQ ID NO:35 GSGYPF 6 LCDR3 chothia SEQ ID NO:36 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 V FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106 SEQ ID NO:37 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213 SEQ ID NO:38 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 Light chain KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT 300 SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH 360 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV 420 HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR 480 EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 540 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 583 SEQ ID NO:39 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213

[0026] The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res.2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1 expression from B DB1 / 154817694.1 22Attorney Docket No.: 116983-5131-WO cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:9).

[0027] The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:10).

[0028] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No.34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:11).

[0029] The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc.2014, DB1 / 154817694.1 23Attorney Docket No.: 116983-5131-WO 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+T cells. Recombinant human IL- 21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No.14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:12).

[0030] When “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the tumor infiltrating lymphocytes (e.g. secondary TILs or genetically modified cytotoxic lymphocytes) described herein may be administered at a dosage of 104to 1011cells / kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to 1010, 106to 1011,107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges. TILs (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The TILs (including, in some cases, genetically engineered TILs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg, et al., New Eng. J. of Med.1988, 319, 1676). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0031] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although DB1 / 154817694.1 24Attorney Docket No.: 116983-5131-WO tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[0032] In some embodiments, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the invention. In some embodiments, the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance.

[0033] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the TILs of the invention.

[0034] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0035] The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of DB1 / 154817694.1 25Attorney Docket No.: 116983-5131-WO completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.

[0036] The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphodepletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variants of the foregoing, are used interchangeably to indicate a chemotherapeutic regimen designed to deplete the patient’s lymphoid immune cells while avoiding depletion of the patient’s myeloid immune cells. Typically, the patient receives a course of non-myeloablative chemotherapy prior to the administration of tumor infiltrating lymphocytes to the patient as described herein.

[0037] The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0038] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual DB1 / 154817694.1 26Attorney Docket No.: 116983-5131-WO inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0039] As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0040] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein. reREP TILs can include for example second expansion TILs or second additional expansion TILs.

[0041] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally DB1 / 154817694.1 27Attorney Docket No.: 116983-5131-WO categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency – for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0042] The term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide.

[0043] The term “RNA” defines a molecule comprising at least one ribonucleotide residue. The term “ribonucleotide” defines a nucleotide with a hydroxyl group at the 2' position of a b-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0044] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in therapeutic compositions of the invention is contemplated. Additional active pharmaceutical DB1 / 154817694.1 28Attorney Docket No.: 116983-5131-WO ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0045] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0046] The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0047] The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof. An “antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding DB1 / 154817694.1 29Attorney Docket No.: 116983-5131-WO portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0048] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

[0049] The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced DB1 / 154817694.1 30Attorney Docket No.: 116983-5131-WO using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0050] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1 domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature, 1989, 341, 544-546), which may consist of a VHor a VLdomain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules known as single chain Fv (scFv); see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In some embodiments, a scFv protein domain comprises a VHportion and a VL portion. A scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VLif the VHdomain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are DB1 / 154817694.1 31Attorney Docket No.: 116983-5131-WO described in U.S. Pat. No.4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991), the disclosures of which are incorporated by reference herein.

[0051] The term “human antibody,” as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0052] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0053] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in DB1 / 154817694.1 32Attorney Docket No.: 116983-5131-WO vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0054] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0055] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0056] The term “human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms “conjugate,” “antibody-drug conjugate”, “ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.

[0057] The terms “humanized antibody,” “humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- DB1 / 154817694.1 33Attorney Docket No.: 116983-5131-WO human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol.1992, 2, 593-596. The antibodies described herein may also be modified to employ any Fc variant which is known to impart an improvement (e.g., reduction) in effector function and / or FcR binding. The Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent Nos.5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated by reference herein.

[0058] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0059] A “diabody” is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448. DB1 / 154817694.1 34Attorney Docket No.: 116983-5131-WO

[0060] The term “glycosylation” refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Patent Nos.5,714,350 and 6,350,861. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see e.g. U.S. Patent Publication No.2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N- acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al., J. Biol. Chem.2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N- DB1 / 154817694.1 35Attorney Docket No.: 116983-5131-WO acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech.1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523.

[0061] “Pegylation” refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No.5,824,778, the disclosures of each of which are incorporated by reference herein.

[0062] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to DB1 / 154817694.1 36Attorney Docket No.: 116983-5131-WO aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, DB1 / 154817694.1 37Attorney Docket No.: 116983-5131-WO and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. II. Cancer Vaccine Manufacturing Processes

[0063] In some aspects the disclosure provides a method for preparing a cancer vaccine, comprising: a) identifying between personalized cancer antigens for a patient; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of the 3-130 personalized cancer antigens; and c) preparing a cancer vaccine in which the total anti-cancer efficacy of the cancer vaccine is maximized (e.g., the predicted total anti-cancer efficacy of the cancer vaccine is maximized) for a given total length of the cancer vaccine.

[0064] Methods for generating cancer vaccines are described in International Application WO2020006242A1 titled “Personalized Cancer Vaccine Epitope Selection”, the content of DB1 / 154817694.1 38Attorney Docket No.: 116983-5131-WO which is herein incorporated by reference in its entirety. The relevant content is reproduced below.

[0065] Methods for generating cancer vaccines according to the disclosure may involve identification of mutations using techniques such as deep nucleic acid or protein sequencing methods as described herein of tissue samples. In some embodiments an initial identification of mutations in a subject’s (e.g., a patient’s) transcriptome is performed. The data from the subject’s (e.g., the patient’s) transcriptome is compared with sequence information from the subject’s (e.g., the patient’s) exome in order to identify patient specific and tumor specific mutations that are expressed. The comparison produces a dataset of putative neoepitopes, referred to as a mutanome. The mutanome may include approximately 100-10,000 candidate mutations per patient. The mutanome is subject to a data probing analysis using a set of inquiries or algorithms to identify an optimal mutation set for generation of a neoantigen vaccine. In some embodiments an mRNA neoantigen vaccine is designed and manufactured. The patient is then treated with the vaccine. In certain embodiments, such a neoantigen- containing vaccine may be a polycistronic vaccine including multiple neoepitopes or one or more single RNA vaccines or a combination thereof.

[0066] In some embodiments the entire method from the initiation of the mutation identification process to the start of patient treatment is achieved in less than 2 months. In other embodiments the whole process is achieved in 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, 3 weeks or less, 2 weeks or less or less than 1 week. In some embodiments the whole method is performed in less than 30 days.

[0067] In a personalized cancer vaccine, the subject specific cancer antigens may be identified in a sample of a patient. The term “biological sample” refers to a sample that contains biological materials such as a DNA, a RNA and a protein. In some embodiments, the biological sample may suitably comprise a bodily fluid from a subject. The bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof. In some embodiments, the sample may be a tissue DB1 / 154817694.1 39Attorney Docket No.: 116983-5131-WO sample or a tumor sample. For instance, a sample of one or more tumor cells may be examined for the presence of subject specific cancer antigens.

[0068] The identification process for specific cancer antigens may involve both transcriptome and exome analysis or only transcriptome or exome analysis. In some embodiments transcriptome analysis is performed first and exome analysis is performed second. The analysis is performed on a biological or tissue sample. In some embodiments a biological or tissue sample is a blood or serum sample. In other embodiments the sample is a tissue bank sample or EBV transformation of B-cells.

[0069] Alternatively the subject specific cancer antigens may be identified in an exosome of the subject. When the antigens for a vaccine are identified in an exosome of the subject, such antigens are said to be representative of exosome antigens of the subject.

[0070] Exosomes are small microvesicles shed by cells, typically having a diameter of approximately 30-100 nm. Exosomes are classically formed from the inward invagination and pinching off of the late endosomal membrane, resulting in the formation of a multivesicular body (MVB) laden with small lipid bilayer vesicles, each of which contains a sample of the parent cell’s cytoplasm. Fusion of the MVB with the cell membrane results in the release of these exosomes from the cell, and their delivery into the blood, urine, cerebrospinal fluid, or other bodily fluids. Exosomes can be recovered from any of these biological fluids for further analysis.

[0071] Nucleic acids within exosomes have a role as bio markers for tumor antigens. An advantage of analyzing exosomes in order to identify subject specific cancer antigens, is that the method circumvents the need for biopsies. This can be particularly advantageous when the patient needs to have several rounds of therapy including identification of cancer antigens, and vaccination.

[0072] A number of methods of isolating exosomes from a biological sample have been described in the art. For example, the following methods can be used: differential centrifugation, low speed centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradients or organelle electrophoresis, magnetic activated cell sorting (MACS), nanomembrane ultrafiltration concentration, Percoll gradient isolation and using microfluidic devices. Exemplary methods are described in US Patent Publication No.2014 / 0212871, for instance. DB1 / 154817694.1 40Attorney Docket No.: 116983-5131-WO

[0073] Once an mRNA vaccine is synthesized, it is administered to the patient. In some embodiments the vaccine is administered on a schedule for up to two months, up to three months, up to four month, up to five months, up to six months, up to seven months, up to eight months, up to nine months, up to ten months, up to eleven months, up to 1 year, up to 1 and ½ years, up to two years, up to three years, or up to four years. The schedule may be the same or varied. In some embodiments the schedule is weekly for the first 3 weeks and then monthly thereafter.

[0074] At any point in the treatment the patient may be examined to determine whether the mutations in the vaccine are still appropriate. Based on that analysis the vaccine may be adjusted or reconfigured to include one or more different mutations or to remove one or more mutations.

[0075] It has been recognized and appreciated that, by analyzing certain properties of cancer associated mutations, optimal neoepitopes may be assessed and / or selected for inclusion in a cancer vaccine. A property of a neoepitope or set of neoepitopes may include, for instance, an assessment of gene or transcript-level expression in patient RNA-seq or other nucleic acid analysis, tissue- specific expression in available databases, known oncogenes / tumor suppressors, variant call confidence score, RNA-seq allele- specific expression, conservative vs. non-conservative AA substitution, position of point mutation (Centering Score for increased TCR engagement), position of point mutation (Anchoring Score for differential HLA binding), Selfness: <100% core epitope homology with patient WES data, HLA-A and - B IC50 for 8mers-l lmers, HLA-DRB1 IC50 for l5mers-20mers, promiscuity Score (e.g., number of patient HLAs predicted to bind), HLA-C IC50for 8mers-11mers, HLA-DRB3-5 IC50 for 15mers-20mers, HLA-DQB1 / A1 IC50 for 15mers-20mers, HLA-DPB1 / A1 IC50 for 15mers-20mers, Class I vs Class II proportion, Diversity of patient HLA-A, -B and DRB1 allotypes covered, proportion of point mutation vs complex epitopes (e.g., frameshifts), and / or pseudo-epitope HLA binding scores.

[0076] In some embodiments, the properties of cancer associated mutations used to identify optimal neoepitopes are properties related to the type of mutation, abundance of mutation in patient sample, immunogenicity, lack of self-reactivity, and nature of peptide composition. The type of mutation should be determined and considered as a factor in determining whether a putative epitope should be included in a vaccine. The type of mutation may vary. In some instances it may be desirable to include multiple different types of mutations in a single DB1 / 154817694.1 41Attorney Docket No.: 116983-5131-WO vaccine. In other instances a single type of mutation may be more desirable. A value for each particular mutation can be weighted and calculated. In some embodiments, a particular mutation is a single nucleotide polymorphism (SNP). In some embodiments, a particular mutation is a complex variant, for example, a peptide sequence resulting from intron retention, complex splicing events, or insertion / deletion mutations changing the reading frame of a sequence.

[0077] The abundance of the mutation in a patient sample may also be scored and factored into the decision of whether a putative epitope should be included in a vaccine. Highly abundant mutations may promote a more robust immune response.

[0078] In some embodiments, the personalized mRNA cancer vaccines described herein may be used for treatment of cancer. As one non-limiting example, the disclosure provides methods for treating a patient having cancer, comprising: a) analyzing a sample derived from the patient is in order to identify one or more personalized cancer antigens; b) determining the anti-tumor efficacy of at least two peptide epitopes for each of the identified personalized cancer antigens; c) preparing a cancer vaccine in which the total anti-cancer efficacy of the cancer vaccine is maximized (e.g., the predicted total anti-cancer efficacy of the cancer vaccine is maximized) for a given total length of the cancer vaccine; and d) administering the cancer vaccine to the patient. Cancer vaccines (e.g., nucleic acid cancer vaccines) may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in cancer or late stage and / or metastatic cancer. In one embodiment, the effective amount of the cancer vaccine (e.g., nucleic acid cancer vaccines) provided to a cell, a tissue or a subject may be enough for immune activation, and in particular antigen specific immune activation.

[0079] In some embodiments, the cancer vaccine (e.g., nucleic acid cancer vaccine) may be administered with an anti-cancer therapeutic agent. The cancer vaccine (e.g., nucleic acid cancer vaccine) and anti-cancer therapeutic can be combined to enhance immune therapeutic responses even further. The cancer vaccine (e.g., nucleic acid cancer vaccines) and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with the cancer vaccine (e.g., nucleic acid cancer vaccine), when the administration of the other therapeutic agents and the cancer DB1 / 154817694.1 42Attorney Docket No.: 116983-5131-WO vaccine (e.g., nucleic acid cancer vaccine) is temporally separated. The separation in time between administrations of these compounds may be a matter of minutes or it may be longer, e.g., hours, days, weeks, months. Other therapeutic agents include but are not limited to anti cancer therapeutic, adjuvants, cytokines, antibodies, antigens, etc.

[0080] In some embodiments, the progression of the cancer can be monitored to identify changes in the expressed antigens. Thus, in some embodiments the method also involves at least one month after the administration of a cancer mRNA vaccine, identifying at least 2 cancer antigens from a sample of the subject to produce a second set of cancer antigens, and administering to the subject a mRNA vaccine having an open reading frame encoding the second set of cancer antigens to the subject. The mRNA vaccine having an open reading frame encoding second set of antigens, in some embodiments, is administered to the subject 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens. In other embodiments the mRNA vaccine having an open reading frame encoding second set of antigens is administered to the subject 1 ½, 2, 2 ½, 3, 3 ½, 4, 4 ½, or 5 years after the mRNA vaccine having an open reading frame encoding the first set of cancer antigens. A. Peptide Epitopes

[0081] The nucleic acid cancer vaccines of the disclosure may encode one or more peptide epitopes (which are portions of personalized cancer antigens). Portions of personalized cancer antigens are segments of personalized cancer antigens that are less than the full-length personalized cancer antigen. A personalized cancer antigen is a tumor-specific antigen, also referred to as a neoantigen that is present in a tumor of an individual that is not expressed or is expressed at low levels in normal non-cancerous tissue of the individual. The antigen may or may not be present in tumors of other individuals.

[0082] In one embodiment, the nucleic acid cancer vaccine is composed of open reading frames that may contain any number of peptide epitopes. In some embodiments the nucleic acid cancer vaccine is composed of open reading frames encoding 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, DB1 / 154817694.1 43Attorney Docket No.: 116983-5131-WO 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 105 or more, 110 or more, 115 or more, 120 or more, 125 or more, 130 or more, 135 or more, 140 or more, 145 or more, 150 or more, 155 or more, 160 or more, 165 or more, 170 or more, 175 or more, 180 or more, 185 or more, 190 or more, 195 or more, 200 or more, 300 or more, 400 or more, 600 or more, 800 or more, or 1,000 or more peptide epitopes. In other embodiments the nucleic acid cancer vaccine is composed of open reading frames encoding 1,000 or less, 800 or less, 600 or less, 400 or less, 300 or less, 200 or less, 195 or less, 190 or less, 185 or less, 180 or less, 175 or less, 170 or less, 165 or less, 160 or less, 155 or less, 150 or less, 145 or less, 140 or less, 135 or less, 130 or less, 125 or less, 120 or less, 115 or less, 110 or less, 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less peptide epitopes. In other embodiments the nucleic acid cancer vaccine is composed of open reading frames encoding up to 1,000, up to 800, up to 600, up to 400, up to 300, up to 200, up to 195, up to 190, up to 185, up to 180, up to 175, up to 170, up to 165, up to 160, up to 155, up to 150, up to 145, up to 140, up to 135, up to 130, up to 125, up to 120, up to 115, up to 110, up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 peptide epitopes, up to 5 peptide epitopes, or up to 3 peptide epitopes.

[0083] In certain embodiments, the nucleic acid cancer vaccine encodes 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70- 80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, 120-130 peptide epitopes, 130-140 peptide epitopes, 140- 150 peptide epitopes, 150-160 peptide epitopes, 160-170 peptide epitopes, 170-180 peptide epitopes, 180-190 peptide epitopes, 190-200 peptide epitopes, 200-300 peptide epitopes, 300- 400 peptide epitopes, 400-500 peptide epitopes, 500-600 peptide epitopes, 600-700 peptide epitopes, 700-800 peptide epitopes, 800-900 peptide epitopes, or 900-1,000 peptide epitopes.

[0084] In certain embodiments, the nucleic acid cancer vaccine encodes 2-200, 5-200, 8-200, 10-200, 2-190, 5-190, 8-190, 10-190, 2-180, 5-180, 8-180, 10-180, 2-170, 5-170, 8-170, 10- 170, 2-160, 5-160, 8-160, 10-160, 2-150, 5-150, 8-150, 10-150, 2-145, 5-145, 8-145, 10-145, DB1 / 154817694.1 44Attorney Docket No.: 116983-5131-WO 2-140, 5-140, 8-140, 10-140, 2-139, 5-139, 8-139, 10-139, 2-138, 5-138, 8-138, 10-138, 2- 137, 5-137, 8-137, 10-137, 2-136, 5-136, 8-136, 10-136, 2-135, 5-135, 8-135, 10-135, 2-134, 5-134, 8-134, 10-134, 2-133, 5-133, 8-133, 10-133, 2-132, 5-132, 8-132, 10-132, 2-131, 5- 131, 8-131, 10-131, 2-130, 5-130, 8-130, 10-130, 2-129, 5-129, 8-129, 10-129, 2-128, 5-128, 8-128, 10-128, 2-127, 5-127, 8-127, 10-127, 2-126, 5-126, 8-126, 10-126, 2-125, 5-125, 8- 125, 10-125, 2-124, 5-124, 8-124, 10-124, 2-123, 5-123, 8-123, 10-123, 2-122, 5-122, 8-122, 10-122, 2-121, 5-121, 8-121, 10-121, 2-120, 5-120, 8-120, 10-120, 2-119, 5-119, 8-119, 10- 119, 2-118, 5-118, 8-118, 10-118, 2-117, 5-117, 8-117, 10-117, 2-116, 5-116, 8-116, 10-116, 2-115, 5-115, 8-115, 10-115, 2-114, 5-114, 8-114, 10-114, 2-113, 5-113, 8-113, 10-113, 2- 112, 5-112, 8-112, 10-112, 2-111, 5-111, 8-111, 10-111, 2-110, 5-110, 8-110, 10-110, 2-100, 5-100, 8-100, or 10-100 peptide epitopes.

[0085] In other embodiments, the nucleic acid cancer vaccine encodes 2-95, 5-95, 8-95, 10- 95, 2-90, 5-90, 8-90, 10-85, 2-85, 5-85, 8-85, 10-85, 2-80, 5-80, 8-80, 10-80, 2-85, 5-85, 8- 85, 10-85, 2-80, 5-80, 8-80, 10-80, 2-75, 5-75, 8-75, 10-75, 2-70, 5-70, 8-70, 10-70, 2-65, 5- 65, 8-65, 10-65, 2-60, 5-60, 8-60, 10-60, 2-55, 5-55, 8-55, 10-55, 2-50, 5-50, 8-50, 10-50, 2- 45, 5-45, 8-45, 10-45, 2-40, 5-40, 8-40, 10-40, 2-39, 5-39, 8-39, 10-39, 2-38, 5-38, 8-38, 10- 38, 2-37, 5-37, 8-37, 10-37, 2-36, 5-36, 8-36, 10-36, 2-35, 5-35, 8-35, 10-35, 2-34, 5-34, 8- 34, 10-34, 2-33, 5-33, 8-33, 10-33, 2-32, 5-32, 8-32, 10-32, 2-31, 5-31, 8-31, 10-31, 2-30, 5- 30, 8-30, 10-30, 2-29, 5-29, 8-29, 10-29, 2-28, 5-28, 8-28, 10-28, 2-27, 5-27, 8-27, 10-27, 2- 26, 5-26, 8-26, 10-26, 2-25, 5-25, 8-25, 10-25, 2-24, 5-24, 8-24, 10-24, 2-23, 5-23, 8-23, 10- 23, 2-22, 5-22, 8-22, 10-22, 2-21, 5-21, 8-21, 10-21, 2-20, 5-20, 8-20, 10-20, 2-19, 5-19, 8- 19, 10-19, 2-18, 5-18, 8-18, 10-18, 2-17, 5-17, 8-17, 10-17, 2-16, 5-16, 8-16, 10-16, 2-15, 5- 15, 8-15, 10-15, 2-14, 5-14, 8-14, 10-14, 2-13, 5-13, 8-13, 10-13, 2-12, 5-12, 8-12, 10-12, 2- 11, 5-11, 8-11, 10-11, 2-10, 5-10, or 8-10 peptide epitopes.

[0086] In yet other embodiments the nucleic acid cancer vaccine encodes 20-200, 30-200, 40-200, 50-200, 20-180, 30-180, 40-180, 50-180, 20-170, 30-170, 40-170, 50-170, 20-160, 30-160, 40-160, 20-150, 30-150, 40-150, 50-150, 20-140, 30-140, 40-140, 50-140, 20-130, 20-130, 40-130, 50-130, 20-120, 30-120, 40-120, 50-120, 20-110, 30-110, 40-110, 50-110, 20-100, 30-100, 40-100, or 50-100 peptide epitopes.

[0087] In some embodiments the nucleic acid cancer vaccines and vaccination methods described herein include open reading frames that encode epitopes or antigens based on DB1 / 154817694.1 45Attorney Docket No.: 116983-5131-WO specific mutations (neoepitopes) and / or those expressed by cancer-germline genes (antigens common to tumors found in multiple patients).

[0088] An epitope, also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells. Epitopes may include B cell epitopes (e.g., predicted B cell reactive epitopes) and T cell epitopes (e.g., predicted T cell reactive epitopes). B-cell epitopes (e.g., predicted B cell reactive epitopes) are peptide sequences which are required for recognition by specific antibody producing B-cells. B cell epitopes (e.g., predicted B cell reactive epitopes) refer to a specific region of the antigen that is recognized by an antibody. T-cell epitopes (e.g., predicted T cell reactive epitopes) are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes (e.g., predicted T cell reactive epitopes) are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I molecules. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous (i.e., may be components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure).

[0089] Each peptide epitope may be any length that is reasonable for an epitope. In some embodiments, the length of each peptide epitope is not necessarily equal. In some embodiments, each peptide epitope in a nucleic acid cancer vaccine is a different length. In certain embodiments, at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and up to and including all) of the peptide epitopes in a nucleic acid cancer vaccine are different lengths.

[0090] In some embodiments, the length of at least one of the peptide epitopes is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at DB1 / 154817694.1 46Attorney Docket No.: 116983-5131-WO least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids. In other embodiments, the length of at least one of the peptide epitopes is 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids. In other embodiments, the length of at least one of the peptide epitopes is up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, or up to 10 amino acids.

[0091] In some embodiments each peptide epitope may be from 5-100 amino acids long (inclusive). In some embodiments the length of at least one of the peptide epitopes is 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 8-100, 8-95, 8-90, 8-85, 8-80, 8-75, 8-70, 8-65, 8-60, 8-55, 8-50, 8-45, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 10-100, 10-95, 10-90, 10-85, 10-80, 10-75, 10-70, 10-65, 10-60, 10-55, 10-50, 10-45, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, or 10-20 amino acids.

[0092] In some embodiments, each of the peptide epitopes encoded by the nucleic acid cancer vaccine may have a different length. In certain embodiments, at least one of the peptide epitopes has a different length than another peptide epitope encoded by the nucleic acid cancer vaccine. Each peptide epitope may be any length that is reasonable for an epitope.

[0093] In some embodiments, different percentages of peptide epitope lengths are encoded by the nucleic acids. All of the percentages described in the following listings may be approximate (i.e ., within 5% of the stated amount). The use of the terms “approximate” and “about” is equivalent.

[0094] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: about 100% < 15 amino acids, about 0% > 15 amino acids; DB1 / 154817694.1 47Attorney Docket No.: 116983-5131-WO about 95% < 15 amino acids, about 5% > 15 amino acids; about 90% < 15 amino acids, about 10% > 15 amino acids; about 85% < 15 amino acids, about 15% > 15 amino acids; about

[0095] 80% < 15 amino acids, about 20% > 15 amino acids; about 75% < 15 amino acids, about 25% > 15 amino acids; about 70% < 15 amino acids, about 30% > 15 amino acids; about 65% < 15 amino acids, about 35% > 15 amino acids; about 60% < 15 amino acids, about 40% > 15 amino acids; about 55% < 15 amino acids, about 45% > 15 amino acids; about 50% < 15 amino acids, about 50% > 15 amino acids; about 45% < 15 amino acids, about 55% > 15 amino acids; about 40% < 15 amino acids, about 60% > 15 amino acids; about 35% < 15 amino acids, about 65% > 15 amino acids; about 30% < 15 amino acids, about 70% > 15 amino acids; about 25% < 15 amino acids, about 75% > 15 amino acids; about 20% < 15 amino acids, about 80% > 15 amino acids; about 15% < 15 amino acids, about 85% > 15 amino acids; about 10% < 15 amino acids, about 90% > 15 amino acids; about 5% < 15 amino acids, about 95% > 15 amino acids; or about 0% < 15 amino acids, about 100% > 15 amino acids.

[0096] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: about 100% < 17 amino acids, about 0% > 17 amino acids; about 95% < 17 amino acids, about 5% > 17 amino acids; about 90% < 17 amino acids, about 10% > 17 amino acids; about 85% < 17 amino acids, about 17% > 17 amino acids; about 80% < 17 amino acids, about 20% > 17 amino acids; about 75% < 17 amino acids, about 25% > 17 amino acids; about 70% < 17 amino acids, about 30% > 17 amino acids; about 65% < 17 amino acids, about 35% > 17 amino acids; about 60% < 17 amino acids, about 40% > 17 amino acids; about 55% < 17 amino acids, about 45% > 17 amino acids; about 50% < 17 amino acids, about 50% > 17 amino acids; about 45% < 17 amino acids, about 55% > 17 amino acids; about 40% < 17 amino acids, about 60% > 17 amino acids; about 35% < 17 amino acids, about 65% > 17 amino acids; about 30% < 17 amino acids, about 70% > 17 amino acids; about 25% < 17 amino acids, about 75% > 17 amino acids; about 20% < 17 amino acids, about 80% > 17 amino acids; about 17% < 17 amino acids, about 85% > 17 amino acids; about 10% < 17 amino acids, about 90% > 17 amino acids; about 5% < 17 amino acids, about 95% > 17 amino acids; or about 0% < 17 amino acids, about 100% > 17 amino acids.

[0097] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: about 100% < 19 amino acids, about 0% > 19 amino acids; DB1 / 154817694.1 48Attorney Docket No.: 116983-5131-WO about 95% < 19 amino acids, about 5% > 19 amino acids; about 90% < 19 amino acids, about 10% > 19 amino acids; about 85% < 19 amino acids, about 19% > 19 amino acids; about 80% < 19 amino acids, about 20% > 19 amino acids; about 75% < 19 amino acids, about 25% > 19 amino acids; about 70% < 19 amino acids, about 30% > 19 amino acids; about 65% < 19 amino acids, about 35% > 19 amino acids; about 60% < 19 amino acids, about 40% > 19 amino acids; about 55% < 19 amino acids, about 45% > 19 amino acids; about 50% < 19 amino acids, about 50% > 19 amino acids; about 45% < 19 amino acids, about 55% > 19 amino acids; about 40% < 19 amino acids, about 60% > 19 amino acids; about 35% < 19 amino acids, about 65% > 19 amino acids; about 30% < 19 amino acids, about 70% > 19 amino acids; about 25% < 19 amino acids, about 75% > 19 amino acids; about 20% < 19 amino acids, about 80% > 19 amino acids; about 19% < 19 amino acids, about 85% > 19 amino acids; about 10% < 19 amino acids, about 90% > 19 amino acids; about 5% < 19 amino acids, about 95% > 19 amino acids; or about 0% < 19 amino acids, about 100% > 19 amino acids.

[0098] In some embodiments, the peptide epitope lengths may be categorized in one of the following groups (for a total of 100%): 8-12 amino acids, 13-17 amino acids, 18-21 amino acids, 22-26 amino acids, or 27-31 amino acids. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acids may be 8-12 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acids may be 13-17 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acids may be 18-21 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acids may be 22-26 amino acids in length. About 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the peptide epitopes encoded by the open reading frames of the nucleic acids may be 27-31 amino acids in length. Several non-limiting examples of the percentages of peptide epitope lengths encoded by the open reading frames of the nucleic acids follow. DB1 / 154817694.1 49Attorney Docket No.: 116983-5131-WO

[0099] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 50% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 50% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 50% 22-26 amino acids, and 50% 27-31 amino acids; 50% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 50% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 50% 22- 26 amino acids, and 0% 27-31 amino acids; 50% 8-12 amino acids, 0% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 50% 22- 26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 50% 13-17 amino acids, 0% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids; or 0% 8-12 amino acids, 0% 13-17 amino acids, 50% 18-21 amino acids, 0% 22-26 amino acids, and 50% 27-31 amino acids.

[0100] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 10% 8-12 amino acids, 40% 13-17 amino acids, 40% 18- 21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 40% 22-26 amino acids, and 0% 27-31 amino acids; 40% 8-12 amino acids, 40% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 10% 8-12 amino acids, 40% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 0% 27-31 amino acids; 40% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 0% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 40% 22-26 amino acids, and 10% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 40% 27-31 amino acids; 0% 8-12 amino acids, 40% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 0% 8-12 amino acids, 10% 13-17 amino acids, 40% 18-21 amino acids, 10% 22-26 amino acids, and 40% 27-31 amino acids; 0% 8-12 amino acids, DB1 / 154817694.1 50Attorney Docket No.: 116983-5131-WO 40% 13-17 amino acids, 10% 18-21 amino acids, 40% 22-26 amino acids, and 10% 27-31 amino acids.

[0101] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 25% 8-12 amino acids, 25% 13-17 amino acids, 25% 18- 21 amino acids, 25% 22-26 amino acids, and 0% 27-31 amino acids; 25% 8-12 amino acids, 25% 13-17 amino acids, 25% 18-21 amino acids, 0% 22-26 amino acids, and 25% 27-31 amino acids; 25% 8-12 amino acids, 25% 13-17 amino acids, 0% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids; 25% 8-12 amino acids, 0% 13-17 amino acids, 25% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids; 0% 8-12 amino acids, 25% 13-17 amino acids, 25% 18-21 amino acids, 25% 22-26 amino acids, and 25% 27-31 amino acids.

[0102] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18- 21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 15% 8- 12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 40% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 40% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 40% 8- 12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids.

[0103] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18- 21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% DB1 / 154817694.1 51Attorney Docket No.: 116983-5131-WO 22-26 amino acids, and 60% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 10% 8- 12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 60% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 60% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 60% 8- 12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids.

[0104] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 15% 8-12 amino acids, 20% 13-17 amino acids, 20% 18- 21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 15% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 20% 22-26 amino acids, and 30% 27-31 amino acids; 20% 8-12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 15% 8-12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 30% 27-31 amino acids; 20% 8- 12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 20% 22-26 amino acids, and 15% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 20% 27-31 amino acids; 30% 8-12 amino acids, 20% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 15% 27-31 amino acids; 30% 8-12 amino acids, 15% 13-17 amino acids, 20% 18-21 amino acids, 15% 22-26 amino acids, and 20% 27-31 amino acids; 30% 8- 12 amino acids, 20% 13-17 amino acids, 15% 18-21 amino acids, 20% 22-26 amino acids, and 15% 27-31 amino acids.

[0105] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 35% 8-12 amino acids, 35% 13-17 amino acids, 10% 18- 21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 35% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 35% 18-21 amino acids, 35% DB1 / 154817694.1 52Attorney Docket No.: 116983-5131-WO 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 35% 27-31 amino acids; 35% 8- 12 amino acids, 10% 13-17 amino acids, 35% 18-21 amino acids, 10% 22-26 amino acids, and 10% 27-31 amino acids; 35% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 35% 8-12 amino acids, 10% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 35% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 35% 22-26 amino acids, and 10% 27-31 amino acids; 10% 8-12 amino acids, 35% 13-17 amino acids, 10% 18-21 amino acids, 10% 22-26 amino acids, and 35% 27-31 amino acids.

[0106] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 30% 8-12 amino acids, 30% 13-17 amino acids, 30% 18- 21 amino acids, 5% 22-26 amino acids, and 5% 27-31 amino acids; 5% 8-12 amino acids, 30% 13-17 amino acids, 30% 18-21 amino acids, 30% 22-26 amino acids, and 5% 27-31 amino acids; 5% 8-12 amino acids, 5% 13-17 amino acids, 30% 18-21 amino acids, 30% 22- 26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 5% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids; 5% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 30% 22- 26 amino acids, and 30% 27-31 amino acids; 5% 8-12 amino acids, 30% 13- 17 amino acids, 30% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids; 30% 8-12 amino acids, 30% 13-17 amino acids, 5% 18-21 amino acids, 30% 22-26 amino acids, and 5% 27-31 amino acids; 30% 8-12 amino acids, 5% 13-17 amino acids, 30% 18-21 amino acids, 5% 22-26 amino acids, and 30% 27-31 amino acids.

[0107] In some embodiments, the percentages of peptide epitope lengths encoded by the nucleic acids may be as follows: 20% 8-12 amino acids, 20% 13-17 amino acids, 20% 18- 21 amino acids, 20% 22-26 amino acids, and 20% 27-31 amino acids.

[0108] In some embodiments, the optimal length of a peptide epitope may be obtained through the following procedure: synthesizing a V5 tag concatemer-test protease site, introducing it into DC cells (for example, using an RNA Squeeze procedure), lysing the cells, and then running an anti-V5 Western blot to assess the cleavage at protease sites.

[0109] The RNA Squeeze technique is an intracellular delivery method by which a variety of materials can be delivered to a broad range of live cells. Cells are subjected to DB1 / 154817694.1 53Attorney Docket No.: 116983-5131-WO microfluidic construction, which causes rapid mechanical deformation. The deformation results in temporary membrane disruption and the newly-formed transient pores. Material is then passively diffused into the cell cytosol via the transient pores. The technique can be used in a variety of cell types, including primary fibroblasts, embryonic stem cells, and a host of immune cells, and has been shown to have relatively high viability in most applications and does not damage sensitive materials, such as quantum dots or proteins, through its actions. Sharei et ah, PNAS (2013); 110(6):2082-7.

[0110] The peptide epitopes described herein may be encoded in any order in the nucleic acid. For example, each of the peptide epitopes may have a length that may be categorized in one of the following groups (for a total of 100%): 8-12 amino acids (represented by “A”), 13-17 amino acids (represented by “B”), 18-21 amino acids (represented by “C”), 22-26 amino acids (represented by “D”), or 27-31 amino acids (represented by “E”). One or more peptide epitopes of any group (e.g ., 8-12 aa) may be encoded consecutively by the nucleic acid (e.g., the nucleic acid may encode two or more peptide epitopes of length “A” in a row and these epitopes may be directly linked or indirectly linked as described elsewhere herein). Additionally, the peptide epitopes of different groups may be interspersed and the nucleic acid may encode epitopes of different groups consecutively (e.g., the nucleic acid may encode a peptide epitope of length A next to a peptide epitope of length B, C, D, or E and these epitopes may be directly linked or indirectly linked as described elsewhere herein).

[0111] As a non-limiting example, the peptide epitopes may be encoded as follows in a nucleic acid or the nucleic acid may encode (at least in part) one of the following combinations of peptide epitopes: DB1 / 154817694.1 54Attorney Docket No.: 116983-5131-WODB1 / 154817694.1 55Attorney Docket No.: 116983-5131-WO

[0112] wherein a peptide epitopes of 8-12 amino acids are represented by “A”, peptide epitopes of 13-17 amino acids are represented by “B”, peptide epitopes of 18-21 amino acids are represented by “C”, peptide epitopes of 22-26 amino acids are represented by “D”, and peptide epitopes of 27-31 amino acids are represented by “E”. DB1 / 154817694.1 56Attorney Docket No.: 116983-5131-WO

[0113] Any of the foregoing combinations of peptide epitopes may be combined. For example, any of the nucleic acid cancer vaccines described herein may encode more than one of the listed groups of peptide epitopes.

[0114] In some embodiments, the peptide epitopes comprise at least one MHC class I epitope and at least one MHC class II epitope. In some embodiments, at least 10% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 20% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 30% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 40% of the peptide epitopes are MHC class I epitopes. In some embodiments, at least 0%, 60%, 70%, 80%, 90%, or 100% of the peptide epitopes are MHC class I epitopes. In some embodiments, none (0%) of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 10% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 20% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 30% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 40% of the peptide epitopes are MHC class II epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or 100% of the peptide epitopes are MHC class II epitopes. In some embodiments, the ratio of MHC class I epitopes to MHC class II epitopes is a ratio selected from about l0%:about 90%; about 20%:about 80%; about 30%:about 70%; about 40%:about 60%; about 50%:about 50%; about 60%:about 40%; about 70%:about 30%; about 80%: about 20%; about 90%: about 10% MHC class 1: MHC class II epitopes. In one embodiment, the ratio of MHC class I : MHC class II epitopes is 1:1. In one embodiment, the ratio of MHC class I : MHC class II epitopes is 2:1. In one embodiment, the ratio of MHC class I : MHC class II epitopes is 3:1. In one embodiment, the ratio of MHC class I : MHC class II epitopes is 4:1. In one embodiment, the ratio of MHC class I : MHC class II epitopes is 5:1. In some embodiments, the ratio of MHC class II epitopes to MHC class I epitopes is a ratio selected from about l0%:about 90%; about 20%:about 80%; about 30%:about 70%; about 40%:about 60%; about 50%:about 50%; about 60%:about 40%; about 70%:about 30%; about 80%: about 20%; about 90%: about 10% MHC class II: MHC class I epitopes. In one embodiment, the ratio of MHC class II : MHC class I epitopes is 1: 1. In one embodiment, the ratio of MHC class II : MHC class I epitopes is 1:2. In one embodiment, the ratio of MHC class II : MHC class I epitopes is 1:3. In one embodiment, the ratio of MHC class II : MHC class I epitopes is 1:4. In one embodiment, the ratio of MHC class II : MHC class I epitopes is 1:5. DB1 / 154817694.1 57Attorney Docket No.: 116983-5131-WO In some embodiments, at least one of the peptide epitopes of the cancer vaccine is a B cell epitope. In some embodiments, one or more predicted T cell reactive epitope of the cancer vaccine comprises between 8-11 amino acids. In some embodiments, one or more predicted B cell reactive epitope of the cancer vaccine comprises between 13-17 amino acids.

[0115] The cancer vaccine of the disclosure, in some aspects comprises an mRNA vaccine encoding multiple peptide epitope antigens arranged with a single amino acid spacer between the peptide epitopes, a short linker between the peptide epitopes, or directly to one another without a spacer between the peptide epitopes. The multiple epitope antigens may include a mixture of MHC class I epitopes and MHC class II epitopes. As a non-limiting example, the multiple peptide epitope antigens may be a polypeptide having the structure:

[0116] where X is an MHC class I epitope of 5-100 amino acids (e.g ., any of the lengths described herein including 8-31 amino acids) in length, Y is an MHC class II epitope of 5- 100 amino acids (e.g., any of the lengths described herein including 8-31 amino acids) in length, and G is glycine.

[0117] The nucleic acid cancer vaccine of the disclosure, in some aspects, comprises a nucleic acid encoding one or more peptide epitopes that include a mutation causing a unique expressed peptide sequence. In some embodiments, a mutation causing a unique expressed peptide sequence may be, but is not limited to, an insertion, deletion, frameshift DB1 / 154817694.1 58Attorney Docket No.: 116983-5131-WO mutation, and / or splicing variant. In some embodiments, the nucleic acid cancer vaccine encodes multiple peptide epitope antigens including one or more single nucleotide polymorphism (SNP) mutations with flanking amino acids on each side of the SNP mutation. In some embodiments, the number of flanking amino acids on each side of the SNP mutation may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30. In some embodiments, the SNP mutation is centrally located and the number of flanking amino acids on each side of the SNP mutation is approximately the same. In other embodiments, the SNP mutation does not have an equivalent number of flanking amino acids on each side. In an embodiment, an epitope of the cancer vaccine comprises an SNP flanked by two Class I sequences, each sequence comprising seven amino acids. In another embodiment, an epitope of the cancer vaccine comprises a SNP flanked by two Class II sequences, each sequence comprising 10 amino acids. In some embodiments, an epitope may comprise a centrally located SNP and flanks which are both Class I sequences, both Class II sequences, or one Class I and one Class II sequence.

[0118] In another embodiment, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of peptide epitopes. Any number of peptide epitopes may be used. In certain embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 5-200 peptide epitopes. In certain embodiments, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 3-130 peptide epitopes. In some embodiments, the concatemeric cancer antigen comprises one or more of: a) the peptide epitopes (e.g., the 3-200 or 3-130 peptide epitopes) are interspersed by cleavage sensitive sites; and / or b) each peptide epitope is linked directly to one another without a linker; and / or c) each peptide epitope is linked to one or another with a single amino acid linker; and / or d) each peptide epitope is linked to one or another with a short linker; and / or e) each peptide epitope comprises 8-31 amino acids and includes one or more SNP mutations (e.g., a centrally located SNP mutation); and / or f) each peptide epitope comprises 8-31 amino acids and includes a mutation causing a unique expressed peptide sequence; and / or g) at least 30% of the peptide epitopes have a highest affinity for class I MHC molecules from a subject; and / or h) at least 30% of the peptide epitopes have a highest affinity for class P MHC molecules from a subject; and / or i) none of the peptide epitopes have a highest affinity for class II MHC molecules from a subject; and / or j) at least 50% of the peptide epitopes have a predicted binding affinity of IC50 <500nM for HLA-A, HLA-B and / or DRB1; and / or k) the DB1 / 154817694.1 59Attorney Docket No.: 116983-5131-WO nucleic acids encoding the peptide epitopes are arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes, 1) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; and / or m) no class II MHC molecules peptide epitopes are present. In some embodiments, peptide epitopes having a “highest affinity” for a class I MHC molecule specifically bind (i.e., bind with greatest affinity) to that class I MHC molecule. In some embodiments, peptide epitopes having a “highest affinity” for a class I MHC molecule have greater binding affinity for that class I MHC molecule than a class II MHC molecule. In some embodiments, peptide epitopes having a “highest affinity” for a class II MHC molecule specifically bind (i.e., bind with greatest affinity) to that class II MHC molecule. In some embodiments, peptide epitopes having a “highest affinity” for a class II MHC molecule have greater binding affinity for that class II MHC molecule than a class I MHC molecule.

[0119] It will be appreciated that a concatemer of 2 or more peptides, e.g., 2 or more neoantigens, may create unintended new epitopes (pseudoepitopes) at peptide boundaries. To prevent or eliminate such pseudoepitopes, class I alleles may be scanned for hits across peptide boundaries in a concatemer. In some embodiments, the peptide order within the concatemer is shuffled to reduce or eliminate pseudoepitope formation. In some embodiments, a linker is used between peptides, e.g., a single amino acid linker such as glycine, to reduce or eliminate pseudoepitope formation. In some embodiments, anchor amino acids can be replaced with other amino acids which will reduce or eliminate pseudoepitope formation. In some embodiments, peptides are trimmed at the peptide boundary within the concatemer to reduce or eliminate pseudoepitope formation.

[0120] In some embodiments the multiple peptide epitope antigens are arranged and ordered to minimize pseudoepitopes. In other embodiments the multiple peptide epitope antigens are a polypeptide that is free of pseudoepitopes. When the cancer antigen epitopes are arranged in a concatemeric structure in a head to tail formation a junction is formed between each of the cancer antigen epitopes. That includes several, i.e., 1-10, amino acids from an epitope on a N-terminus of the peptide and several, i.e., 1-10, amino acids on a C- terminus of an adjacent directly linked epitope. It is important that the junction not be an immunogenic peptide that may produce an immune response. In some embodiments the junction forms a peptide sequence that binds to an HLA protein of a subject for which the personalized cancer vaccine is designed with an IC50 greater than about 50 nM. In other DB1 / 154817694.1 60Attorney Docket No.: 116983-5131-WO embodiments the junction peptide sequence binds to an HLA protein of a subject with an IC50 greater than about 10 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nm, or 500 nM. B. Personalized Cancer Vaccines

[0121] In some aspects, the present disclosure provides a nucleic acid cancer vaccine comprising one or more nucleic acids, wherein each of the nucleic acids encodes at least one suitable cancer antigen such as a personalized antigen specific for a cancer subject. For instance, the nucleic acid cancer vaccine may include nucleic acids encoding one or more cancer antigens specific for each subject, referred to as neoepitopes. Antigens that are expressed in or by tumor cells are referred to as “tumor associated antigens.” A particular tumor associated antigen may or may not also be expressed in non-cancerous cells. Many tumor mutations are well known in the art. Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced in comparison to that in cancerous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes. Neoepitopes are completely foreign to the body and thus would not produce an immune response against healthy tissue or be masked by the protective components of the immune system. In some embodiments personalized vaccines based on neoepitopes are desirable because such vaccine formulations will maximize specificity against a patient’s specific tumor. Mutation-derived neoepitopes can arise from point mutations, non-synonymous mutations leading to different amino acids in the protein; read-through mutations in which a stop codon is modified or deleted, leading to translation of a longer protein with a novel tumor-specific sequence at the C-terminus; splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor-specific protein sequence; chromosomal rearrangements that give rise to a chimeric protein with tumor-specific sequences at the junction of 2 proteins (i.e., gene fusion); frameshift mutations or deletions that lead to a new open reading frame with a novel tumor-specific protein sequence; and / or translocations.

[0122] Methods for generating personalized cancer vaccines generally involve identification of mutations, e.g., using deep nucleic acid or protein sequencing techniques, identification of neoepitopes, e.g., using application of validated peptide-MHC binding prediction algorithms or other analytical techniques to generate a set of candidate T cell epitopes that may bind to patient HLA alleles and are based on mutations present in tumors, DB1 / 154817694.1 61Attorney Docket No.: 116983-5131-WO optional demonstration of antigen-specific T cells against selected neoepitopes or demonstration that a candidate neoepitope is bound to HLA proteins on the tumor surface and development of the vaccine. Examples of techniques for identifying mutations include but are not limited to dynamic allele- specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide- specific ligation, the TaqMan system as well as various DNA “chip” technologies (e.g.., Affymetrix SNP chips), and methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle amplification.

[0123] Several deep nucleic acid and protein sequencing techniques are known in the art. Any type of sequence analysis method can be used. For instance nucleic acid sequencing may be performed on whole tumor genomes, tumor exomes (protein-encoding DNA), and / or tumor transcriptomes. Real-time single molecule sequencing-by-synthesis technologies rely on the detection of fluorescent nucleotides as they are incorporated into a nascent strand of DNA that is complementary to the template being sequenced. Other rapid high throughput sequencing methods also exist. Protein sequencing may be performed on tumor proteomes. Additionally, protein mass spectrometry may be used to identify or validate the presence of mutated peptides bound to MHC proteins on tumor cells. Peptides can be acid-eluted from tumor cells or from HLA molecules that are immunoprecipitated from tumors, and then identified using mass spectrometry. The results of the sequencing may be compared with known control sets or with sequencing analysis performed on normal tissue of the patient. In some embodiments, these neoepitopes bind to class I HLA proteins with a greater affinity than the wild-type peptide and / or are capable of activating anti-tumor CD8 T-cells. Identical mutations in any particular gene are rarely found across tumors.

[0124] Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells. The proteins of MHC class I are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells, and are then presented to cytotoxic T-lymphocytes (CTLs). T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules of MHC class I. Each cytotoxic T-lymphocyte expresses a unique T-cell receptor which is capable of binding specific MHC / peptide complexes.

[0125] Using computer algorithms, it is possible to predict potential neoepitopes such as putative T-cell reactive epitopes, i.e., peptide sequences, which are bound by the MHC DB1 / 154817694.1 62Attorney Docket No.: 116983-5131-WO molecules of class I or class II in the form of a peptide-presenting complex and then, in this form, recognized by the T-cell receptors of T-lymphocytes. Examples of programs useful for identifying peptides which will bind to MHC include, for instance: Lonza Epibase, SYFPEITHI (Rammensee et ah, Immunogenetics, 50 (1999), 213-219) and HLA_BIND (Parker et ah, J. Immunol., 152 (1994), 163-175).

[0126] Once putative neoepitopes are selected, they can be further tested using in vitro and / or in vivo assays. Conventional in vitro lab assays, such as Elispot assays, may be used with an isolate from each patient to refine the list of neoepitopes selected based on the algorithm’s predictions.

[0127] In some embodiments the nucleic acid cancer vaccines and vaccination methods described herein may include peptide epitopes or antigens based on specific mutations (neoepitopes) and those expressed by cancer-germline genes (antigens common to tumors found in multiple patients, referred to herein as “traditional cancer antigens” or “shared cancer antigens”). In some embodiments, a traditional antigen is one that is known to be found in cancers or tumors generally or in a specific type of cancer or tumor. In some embodiments, a traditional cancer antigen is a non-mutated tumor antigen. In some embodiments, a traditional cancer antigen is a mutated tumor antigen.

[0128] In some embodiments, the nucleic acid cancer vaccines and methods described herein may include peptide epitopes based on cancer / testis (CT) antigens. Cancer / testis antigen expression is limited to male germ cells in healthy adults, but ectopic expression has been observed in tumor cells of multiple types of human cancer. Since male germ cells are devoid of HLA-class I molecules and cannot present antigens to T cells, cancer / testis antigens are generally considered neoantigens when expressed in cancer cells and have the capacity to elicit immune responses that are strictly cancer- specific. Cancer / testis antigens for use with the compositions and methods described herein may be any such cancer / testis antigen known in the field including, but not limited to, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, GAGE1, GAGE2A, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE8, SSX1, SSX2, SSX2b, SSX3, SSX4, CTAG1B, LAGE-lb, CTAG2, MAGEC1, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, DB1 / 154817694.1 63Attorney Docket No.: 116983-5131-WO XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1, SPOl l, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, HORMAD1, HORMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, CT47B1, SLC06A1, TAG, LEMD1, HSPB9, CCDC110, ZNF165, SPACA3, CXorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993, PASD1, LOC647107, TULP2, CT66 / AA884595, PRSS54, RBM46, CT69 / BC040308, CT70 / BI818097, SPINLW1, TSSK6, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf6l, PBK, C2lorf99, OIP5, CEP290, CABYR, SPAG9, MPHOSPH1, ROPN1, PLAC1, CALR3, PRM1, PRM2, CAGE1, TTK, LY6K, IMP-3, AKAP4, DPPA2, KIAA0100, DCAF12, SEMG1, POTED, POTEE, POTEA, POTEG, POTEB, POTEC, POTEH, GOLGAGL2 FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBX039, RGS22, cyclin Al, Cl5orf60, CCDC83, TEKT5, NR6A1, TMPRSS12, TPPP2, PRSS55, DMRT1, EDAG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE12B, GAGE12C, GAGE12D, GAGE12E, GAGE12F, GAGE12G, GAGE12H, GAGE 121, GAGE12J, GAGE13, LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, XAGE1E, XAGE2B / CTD-2267G17.3, and / or variants thereof.

[0129] In some embodiments, the nucleic acid cancer vaccines may further include one or more nucleic acids encoding for one or more non-mutated tumor antigens. In some embodiments, the nucleic acid cancer vaccines may further include one or more nucleic acids encoding for one or more mutated tumor antigens.

[0130] Many tumor antigens are known in the art. Cancer or tumor antigens (e.g., traditional cancer antigens) for use with the compositions and methods described herein may DB1 / 154817694.1 64Attorney Docket No.: 116983-5131-WO be any such cancer or tumor antigens known in the field. In some embodiments, the cancer or tumor antigen (e.g., the traditional cancer antigen) is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4- IBB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin anb , LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin- 4, NKGD2, NOTCH, 0X40, OX40L, PD-l, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-l, TACI, TAG-72, Tenascin, TIM3, TRAILR1 , TRAILR2,VEGFR- 1 , VEGFR-2, VEGFR-3, and / or variants thereof.

[0131] Epitopes can be identified using a free or commercial database (Lonza Epibase, antitope for example). Such tools are useful for predicting the most immunogenic epitopes within a target antigen protein. The selected peptides may then be synthesized and screened in human HLA panels, and the most immunogenic sequences are used to construct the nucleic acids encoding the peptide epitope(s). One strategy for mapping epitopes of Cytotoxic T-Cells based on generating equimolar mixtures of the four C-terminal peptides for each nominal 1 l-mer across a protein. This strategy would produce a library antigen containing all the possible active CTL epitopes.

[0132] The neoepitopes may be designed to optimally bind to MHC in order to promote a robust immune response. In some embodiments each peptide epitope comprises an antigenic region and a MHC stabilizing region. An MHC stabilizing region is a sequence which stabilizes the peptide in the MHC.

[0133] All of the MHC stabilizing regions within the epitopes may be the same or they may be different. The MHC stabilizing regions may be at the N terminal portion of the peptide or the C terminal portion of the peptide. Alternatively the MHC stabilizing regions may be in the central region of the peptide.

[0134] The MHC stabilizing region may be 5-10, 5-15, 8-10, 1-5, 3-7, or 3-8 amino acids in length. In yet other embodiments the antigenic region is 5-100 amino acids in length. The peptides interact with the molecules of MHC class I by competitive affinity binding within the endoplasmic reticulum, before they are presented on the cell surface. The affinity DB1 / 154817694.1 65Attorney Docket No.: 116983-5131-WO of an individual peptide is directly linked to its amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence. The peptide being presented in the MHC is held by the floor of the peptide-binding groove, in the central region of the al / a2 heterodimer (a molecule composed of two non-identical subunits). The sequence of residues of the peptide-binding groove’s floor determines which particular peptide residues it binds.

[0135] Optimal binding regions may be identified by a computer assisted comparison of the affinity of a binding site (MHC pocket) for a particular amino acid at each amino acid in the binding site for each of the target epitopes to identify an ideal binder for all of the examined antigens. The MHC stabilization regions of the epitopes may be identified using amino acid prediction matrices of data points for a binding site. An amino acid prediction matrix is a table having a first and a second axis defining data points. Prediction matrices can be generated as shown in Singh, H. and Raghava, G.P.S. (2001), “ProPred: prediction of HLA- DR binding sites.” Bioinformatics, 17(12), 1236-37). In some embodiments, the prediction matrix is based on evolutionary conservation, in another embodiment, the prediction matrix uses physiochemical similarity to examine how similar a somatic amino acid is to the germline amino acid (e.g., Kim et ah, J Immunol.2017: 3360-3368). The similarity of the somatic amino acid to the germline amino acid approximates how a mutation affects binding (e.g., T cell receptor recognition). In some embodiments, less similarity is indicative of improved binding (e.g., T cell receptor recognition).

[0136] In some embodiments the MHC stabilizing region is designed based on the subject’s particular MHC. In that way the MHC stabilizing region can be optimized for each patient.

[0137] The neoepitopes selected for inclusion in the cancer vaccine (e.g., nucleic acid cancer vaccine) will typically be high affinity binding peptides. In some aspect the neoepitope binds an HLA protein with greater affinity than a wild-type peptide. The neoepitope has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less in some embodiments. Typically, peptides with predicted IC50 <50 nM, are generally considered medium to high affinity binding peptides and will be selected for testing their affinity empirically using biochemical assays of HLA-binding. Finally, it will be determined whether the human immune system can mount effective DB1 / 154817694.1 66Attorney Docket No.: 116983-5131-WO immune responses against these mutated tumor antigens and thus effectively kill tumor but not normal cells.

[0138] In some embodiments, the neoepitopes are 13 residues or less in length and may consist of between about 8 and about 11 residues, particularly 9 or 10 residues. In other embodiments the neoepitopes may be designed to be longer. For instance, the neoepitopes may have extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product. The use of a longer peptide may allow endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses.

[0139] Neoepitopes having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell or B cell. For instance, the neoepitopes may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and / or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be probed using D- amino acids. Such modifications may be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp.1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

[0140] The neoepitopes can also be modified by extending or decreasing the compound’s amino acid sequence, e.g., by the addition or deletion of amino acids. The peptides, polypeptides or analogs can also be modified by altering the order or composition of certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity.

[0141] Typically, a series of peptides with single amino acid substitutions are employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For DB1 / 154817694.1 67Attorney Docket No.: 116983-5131-WO instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell or B cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo-oligomers or hetero-oligomers. The number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.

[0142] The neoepitopes may also comprise isosteres of two or more residues in the neoepitopes. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the alpha-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed„ 1983).

[0143] The consideration of immunogenicity is an important component in the selection of optimal neoepitopes for inclusion in a vaccine. As a set of non-limiting examples, immunogenicity may be assessed by analyzing the MHC binding capacity of a neoepitope, HLA promiscuity, mutation position, predicted T cell reactivity, actual T cell reactivity, structure leading to particular conformations and resultant solvent exposure, and representation of specific amino acids. Known algorithms such as the NetMHC prediction algorithm can be used to predict capacity of a peptide to bind to common HLA-A and -B alleles. In some embodiments, the NetMHC prediction algorithm uses the IC50 to determine binding capacity. In other embodiments, the NetMHC prediction algorithm uses percent rank and eluted ligand data to determine binding capacity (Jurtz et al., J Immunol.2017 Nov l;l99(9):3360-3368). The percent rank method results in a more balanced distribution of DB1 / 154817694.1 68Attorney Docket No.: 116983-5131-WO predicted binders across different HLA alleles. Structural assessment of a MHC bound peptide may also be conducted by in silico 3 -dimensional analysis and / or protein docking programs. Use of a predicted epitope structure when bound to a MHC molecule, such as acquired from a Rosetta algorithm, may be used to evaluate the degree of solvent exposure of an amino acid residues of an epitope when the epitope is bound to a MHC molecule. T cell reactivity may be assessed experimentally with epitopes and T cells in vitro. Alternatively T cell reactivity may be assessed using T cell response / sequence datasets.

[0144] One important aspect of a neoepitope included in a vaccine is a lack of self- reactivity. The putative neoepitopes may be screened to confirm that the epitope is restricted to tumor tissue, for instance, arising as a result of genetic change within malignant cells. Ideally, the epitope should not be present in normal tissue of the patient and thus, self-similar epitopes are filtered out of the dataset. A personalized coding genome may be used as a reference for comparison of neoantigen candidates to determine lack of self-reactivity. In some embodiments, a personalized coding genome is generated from an individualized transcriptome and / or exome.

[0145] The nature of peptide composition may also be considered in the epitope design. For instance a score can be provided for each putative epitope on the value of conserved versus non-conserved amino acids found in the epitope.

[0146] In some embodiments, the analysis performed by the tools described herein may include comparing different sets of properties acquired at different times from a patient, i.e., prior to and following a therapeutic intervention, from different tissue samples, from different patients having similar tumors, etc. In some embodiments, an average of peak values from one set of properties may be compared with an average of peak values from another set of properties. For example, an average value for HLA binding may be compared between two different sets of distributions. The two sets of distributions may be determined for time durations separated by days, months, or years, for instance. C. Nucleic Acids / Polynucleotides

[0147] Cancer vaccines (e.g., nucleic acid cancer vaccines), as provided herein, comprise at least one (one or more) nucleic acid having an open reading frame encoding at least one peptide epitope. The term “nucleic acid,” in its broadest sense, includes any DB1 / 154817694.1 69Attorney Docket No.: 116983-5131-WO compound and / or substance that comprises a polymer of nucleotides. These polymers are also referred to as polynucleotides.

[0148] Nucleic acids may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a b- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino- LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

[0149] As a non-limiting example, when a DNA nucleic acid cancer vaccine as described herein is delivered to a cell, the DNA is transcribed into RNA, and the RNA will be processed into a polypeptide by the intracellular machinery which can then process the polypeptide into immunosensitive fragments capable of stimulating an immune response against a tumor or population of cancerous cells. As a non-limiting example, when an RNA (e.g., mRNA) nucleic acid cancer vaccine as described herein is delivered to a cell, the RNA (e.g., mRNA) will be processed into a polypeptide by the intracellular machinery which can then process the polypeptide into immunosensitive fragments capable of stimulating an immune response against a tumor or population of cancerous cells.

[0150] In some embodiments, nucleic acids of the present disclosure function as messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to any nucleic acid that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

[0151] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Nucleic acids of the present disclosure may function as mRNA but can be distinguished from wild- type mRNA in their functional and / or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

[0152] Polynucleotides of the present disclosure, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies DB1 / 154817694.1 70Attorney Docket No.: 116983-5131-WO in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0153] In some embodiments, a codon optimized sequence shares less than 95% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild- type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon optimized sequence shares less than 90% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon optimized sequence shares less than 85% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon optimized sequence shares less than 80% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon optimized sequence shares less than 75% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide).

[0154] In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally- DB1 / 154817694.1 71Attorney Docket No.: 116983-5131-WO occurring or wild- type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity with a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide).

[0155] In some embodiments a codon optimized RNA may, for instance, be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA. D. Antigens / Antigenic Polypeptides

[0156] In some embodiments, each peptide epitope may be from 5-100 amino acids long (inclusive). In some embodiments the length of at least one of the peptide epitopes is 5- 100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 8-100, 8-95, 8-90, 8-85, 8-80, 8-75, 8-70, 8-65, 8-60, 8-55, 8-50, 8-45, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 10-100, 10-95, 10-90, 10-85, 10-80, 10-75, 10-70, 10-65, 10-60, 10-55, 10-50, 10-45, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, or 10-20 amino acids.

[0157] In some embodiments, each of the peptide epitopes encoded by the nucleic acid cancer vaccine may have a different length. In certain embodiments, at least one of the peptide epitopes has a different length than another peptide epitope encoded by the nucleic acid cancer vaccine. Each peptide epitope may be any length that is reasonable for an epitope.

[0158] Polypeptides for use with the instant disclosure include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may DB1 / 154817694.1 72Attorney Docket No.: 116983-5131-WO be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.

[0159] The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a native or reference sequence.

[0160] In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” is one which contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and / or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.

[0161] “Orthologs” refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

[0162] “Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations including, for example, substitutions, additions, or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

[0163] The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used synonymously with the term “variant” but generally refers to a molecule DB1 / 154817694.1 73Attorney Docket No.: 116983-5131-WO that has been modified and / or changed in any way relative to a reference molecule or starting molecule.

[0164] As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends).

[0165] Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

[0166] “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0167] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. DB1 / 154817694.1 74Attorney Docket No.: 116983-5131-WO

[0168] “Features” when referring to polypeptide or polynucleotide are defined as distinct amino acid sequence-based or nucleotide -based components of a molecule respectively. Features of the polypeptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.

[0169] As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0170] As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and“amino acid side chain.” As used herein when referring to polynucleotides the terms “site” as it pertains to nucleotide based embodiments is used synonymously with “nucleotide.” A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide or polynucleotide based molecules.

[0171] As used herein the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non- polypeptide based moiety such as an organic conjugate.

[0172] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any protein that includes a stretch of 10, DB1 / 154817694.1 75Attorney Docket No.: 116983-5131-WO 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65%, or 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

[0173] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or“identity” with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides (e.g., DNA molecules and / or RNA molecules), as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “Percent identity” or “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non identical sequences can be disregarded DB1 / 154817694.1 76Attorney Docket No.: 116983-5131-WO for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0174] Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in DB1 / 154817694.1 77Attorney Docket No.: 116983-5131-WO publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Stephen F. Altschul, et al (1997), “Gapped BEAST and PSTBFAST: a new generation of protein database search programs”, Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol.147:195- 197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970)“A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0175] As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are termed homologous. Homology is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4- DB1 / 154817694.1 78Attorney Docket No.: 116983-5131-WO 5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

[0176] Homology implies that the compared sequences diverged in evolution from a common origin. The term “homolog” refers to a first amino acid sequence or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The term “homolog” may apply to the relationship between genes and / or proteins separated by the event of speciation or to the relationship between genes and / or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one. E. Chemical Modifications 1. Modified Nucleotide Sequences Encoding Epitope Antigen Polypeptides

[0177] In some embodiments, the nucleic acid cancer vaccine of the invention comprises one or more chemically modified nucleobases. The invention includes modified polynucleotides comprising a polynucleotide described herein (e.g., a nucleic acid comprising a nucleotide sequence encoding one or more cancer peptide epitopes). The modified nucleic acids can be chemically modified and / or structurally modified. When the nucleic acids of the present invention are chemically and / or structurally modified the polynucleotides can be referred to as “modified nucleic acids.”

[0178] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding one or more cancer peptide epitopes. A“nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). A“nucleotide” refers to a nucleoside including a phosphate group. Modified nucleotides can by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. DB1 / 154817694.1 79Attorney Docket No.: 116983-5131-WO Nucleic acids can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. The modified nucleic acids disclosed herein can comprise various distinct modifications. In some embodiments, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide introduced to a cell can exhibit one or more desirable properties such as, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.

[0179] In some embodiments, a nucleic acid disclosed herein (e.g., a nucleic acid encoding one or more peptide epitopes) is structurally modified. As used herein, a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” can be chemically modified to “AT-5meC-G.” The same polynucleotide can be structurally modified from “ATCG” to “ATCCCG.” Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the nucleic acid.

[0180] In some embodiments, the nucleic acids of the instant disclosure are chemically modified. As used herein in reference to a nucleic acid, the terms“chemical modification” or, as appropriate,“chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percentage, or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '- terminal mRNA cap moieties.

[0181] In some embodiments, the nucleic acids of the instant disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are DB1 / 154817694.1 80Attorney Docket No.: 116983-5131-WO replaced by a uridine analog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment, the polynucleotides can have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way).

[0182] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non- standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into polynucleotides of the present disclosure.

[0183] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA, the “T”s would be substituted for“U”s.

[0184] Cancer vaccines of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one (e.g., 3-200 or 3-130) peptide epitope(s), wherein the nucleic acid comprises nucleotides and / or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally- occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and / or nucleoside as are recognized in the art.

[0185] In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0186] In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non- DB1 / 154817694.1 81Attorney Docket No.: 116983-5131-WO limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT / US2012 / 058519; PCT / US2013 / 075177; PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413; PCT / US2015 / 36773; PCT / US2015 / 36759; PCT / US2015 / 36771; or PCT / IB 2017 / 051367 all of which are incorporated by reference herein for this purpose.

[0187] Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

[0188] Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0189] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0190] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0191] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on intemucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

[0192] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., DNA nucleic acids or RNA nucleic acids, such as mRNA nucleic acids). A DB1 / 154817694.1 82Attorney Docket No.: 116983-5131-WO “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

[0193] Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into nucleic acids of the present disclosure.

[0194] In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1 -methyl-pseudouridine (hiΐy), 1 - ethyl- pseudouridine (eΐy), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, l-methoxymethyl pseudouridine, 5-methyl cytidine, and / or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0195] In some embodiments, a RNA nucleic acid of the disclosure comprises 1 - methyl- pseudouridine (m 1 y) substitutions at one or more or all uridine positions of the nucleic acid. DB1 / 154817694.1 83Attorney Docket No.: 116983-5131-WO

[0196] In some embodiments, a RNA nucleic acid of the disclosure comprises 1 - methyl- pseudouridine (m 1 y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0197] In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.

[0198] In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0199] In some embodiments, a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

[0200] In some embodiments, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1- methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1 -methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0201] The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly-A tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0202] The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U, or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, DB1 / 154817694.1 84Attorney Docket No.: 116983-5131-WO from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0203] The nucleic acids may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0204] In some embodiments, the nucleic acid can include any useful linker between the nucleosides. Such linkers, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3'-alkylene phosphonates, 3 '-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH2-O- N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, morpholino linkages, -N(CH3)-CH2-CH2-, oligonucleosides DB1 / 154817694.1 85Attorney Docket No.: 116983-5131-WO with heteroatom internucleoside linkage, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate intemucleoside linkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

[0205] The modified nucleosides and nucleotides (e.g., building block molecules), which can be incorporated into a nucleic acid (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C1-6alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8cycloalkyl; optionally substituted C3-8cycloalkoxy; optionally substituted C6-10aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), - O(CH2CH2O)n CH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2'-hydroxyl is connected by a C1-6alkylene or C1-6heteroalkylene bridge to the 4 '-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0206] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and“unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace DB1 / 154817694.1 86Attorney Docket No.: 116983-5131-WO with a-L- threofuranosyl-(3' 2')) , and peptide nucleic acid (PNA, where 2-amino-ethyl- glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar. Such sugar modifications are described in, for example, International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entireties for this purpose.

[0207] The nucleic acids of the disclosure (e.g., a nucleic acid encoding one or more peptide epitopes or a functional fragment or variant thereof) can include a combination of modifications to the sugar, the nucleobase, and / or the intemucleoside linkage. These combinations can include any one or more modifications described herein.

[0208] The nucleic acid cancer vaccines disclosed herein are compositions, including pharmaceutical compositions. The disclosure also encompasses methods for the selection, design, preparation, manufacture, formulation, and / or use of nucleic acid cancer vaccines as provided herein. Also provided are systems (e.g., computerized systems), processes, devices and kits for the selection, design, and / or utilization of the nucleic acid cancer vaccines described herein. F. In Vitro Transcription of RNA (e.g., mRNA)

[0209] Cancer vaccines of the present disclosure may comprise at least one nucleic acid (e.g., an RNA polynucleotide, such as an mRNA (message RNA) or an mmRNA (modified mRNA)). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “m vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a poly-A tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0210] In some embodiments, a nucleic acid includes 15 to 3,000 nucleotides. For example, a polynucleotide may include 15 to 50, 15 to 100, 15 to 200, 15 to 300, 15 to 400, 15 to 500, 15 to 600, 15 to 700, 15 to 800, 15 to 900, 15 to 1000, 15 to 1200, 15 to 1400, 15 to 1500, 15 to 1800, 15 to 2000, 15 to 2500, 15 to 3000, 50 to 100, 50 to 200, 50 to 300, 50 to DB1 / 154817694.1 87Attorney Docket No.: 116983-5131-WO 400, 50 to 500, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 50 to 1200, 50 to 1400, 50 to 1500, 50 to 1800, 50 to 2000, 50 to 2500, 50 to 3000, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 100 to 600, 100 to 700, 100 to 800, 100 to 900, 100 to 1000, 100 to 1200, 100 to 1400, 100 to 1500, 100 to 1800, 100 to 2000, 100 to 2500, 100 to 3000, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200, to 800, 200 to 900, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 2500, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 2500, 1000 to 3000, 1500 to 3000, 2500 to 3000, or 2000 to 3000 nucleotides).

[0211] In other aspects, the disclosure relates to a method for preparing a nucleic acid cancer vaccine (e.g., an mRNA cancer vaccine) by IVT methods. In vitro transcription (IVT) methods permit template-directed synthesis of RNA molecules of almost any sequence. The size of the RNA molecules that can be synthesized using IVT methods range from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods permit synthesis of large quantities of RNA transcript (e.g., from microgram to milligram quantities). See Beckert el al, Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703:29-41(2011); Rio et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.; Cooper, Geoffery M. The Cell: A Molecular Approach.4th ed. Washington D.C.: ASM Press, 2007.262-299, each of which is herein incorporated by reference for this purpose. Generally, IVT utilizes a DNA template featuring a promoter sequence upstream of a sequence of interest. The promoter sequence is most commonly of bacteriophage origin (e.g., the T7, T3 or SP6 promoter sequence) but many other promotor sequences can be tolerated including those designed de novo. Transcription of the DNA template is typically best achieved by using the RNA polymerase corresponding to the specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT is generally initiated at a dsDNA but can proceed on a single strand.

[0212] It will be appreciated that nucleic acid cancer vaccines (e.g., mRNA cancer vaccines) of the present disclosure, e.g., mRNAs encoding the cancer antigen, may be made using any appropriate synthesis method. For example, in some embodiments, mRNA vaccines of the present disclosure are made using IVT from a single bottom strand DNA as a template and complementary oligonucleotide that serves as promotor. The single bottom strand DNA may act as a DNA template for in vitro transcription of RNA, and may be DB1 / 154817694.1 88Attorney Docket No.: 116983-5131-WO obtained from, for example, a plasmid, a PCR product, or chemical synthesis. In some embodiments, the single bottom strand DNA is linearized from a circular template. The single bottom strand DNA template generally includes a promoter sequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods of making RNA using a single bottom strand DNA and a top strand promoter complementary oligonucleotide are known in the art. An exemplary method includes, but is not limited to, annealing the DNA bottom strand template with the top strand promoter complementary oligonucleotide (e.g., T7 promoter complementary oligonucleotide, T3 promoter complementary oligonucleotide, or SP6 promoter complementary oligonucleotide), followed by IVT using an RNA polymerase corresponding to the promoter sequence, e.g., aT7 RNA polymerase, a T3 RNA polymerase, or an SP6 RNA polymerase.

[0213] IVT methods can also be performed using a double- stranded DNA template. For example, in some embodiments, the double-stranded DNA template is made by extending a complementary oligonucleotide to generate a complementary DNA strand using strand extension techniques available in the art. In some embodiments, a single bottom strand DNA template containing a promoter sequence and sequence encoding one or more peptide epitopes of interest is annealed to a top strand promoter complementary oligonucleotide and subjected to a PCR-like process to extend the top strand to generate a double-stranded DNA template. Alternatively or additionally, a top strand DNA containing a sequence complementary to the bottom strand promoter sequence and complementary to the sequence encoding one or more peptide epitopes of interest is annealed to a bottom strand promoter oligonucleotide and subjected to a PCR-like process to extend the bottom strand to generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. In some embodiments, a double-stranded DNA template is synthesized wholly or in part by chemical synthesis methods. The double- stranded DNA template can be subjected to in vitro transcription as described herein.

[0214] In another aspect, nucleic acid cancer vaccines of the present disclosure comprising, e.g., mRNAs encoding the peptide epitopes, may be made using two DNA strands that are complementary across an overlapping portion of their sequence, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions are annealed. These single- stranded overhangs can be made double-stranded by extending using the other strand as a template, thereby generating double- stranded DNA. In some cases, this primer DB1 / 154817694.1 89Attorney Docket No.: 116983-5131-WO extension method can permit larger ORFs to be incorporated into the template DNA sequence, e.g., as compared to sizes incorporated into the template DNA sequences obtained by top strand DNA synthesis methods. In the primer extension method, a portion of the 3 '- end of a first strand (in the 5 '-3' direction) is complementary to a portion the 3 '-end of a second strand (in the 3’-5' direction). In some such embodiments, the single first strand DNA may include a sequence of a promoter (e.g., T7, T3, or SP6), optionally a 5'-UTR, and some or all of an ORF (e.g., a portion of the 5 '-end of the ORF). In some embodiments, the single second strand DNA may include complementary sequences for some or all of an ORF (e.g., a portion complementary to the 3 '-end of the ORF), and optionally a 3'-UTR, a stop sequence, and / or a poly-A tail. Methods of making RNA using two synthetic DNA strands may include annealing the two strands with overlapping complementary portions, followed by primer extension using one or more PCR-like cycles to extend the strands to generate a double- stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. Such double- stranded DNA can be subjected to in vitro transcription as described herein.

[0215] In another aspect, nucleic acid vaccines of the present disclosure comprising, e.g., mRNAs encoding the peptide epitopes, may be made using synthetic double-stranded linear DNA molecules, such as gBlocks®(Integrated DNA Technologies, Coralville, Iowa), as the double-stranded DNA template. An advantage to such synthetic double- stranded linear DNA molecules is that they provide a longer template from which to generate mRNAs. For example, gBlocks®can range in size from 45-1000 (e.g., 125-750 nucleotides). In some embodiments, a synthetic double-stranded linear DNA template includes a full length 5'- UTR, a full length 3'-UTR, or both. A full length 5'-UTR may be up to 100 nucleotides in length, e.g., about 40-60 nucleotides. A full length 3'-UTR may be up to 300 nucleotides in length, e.g., about 100-150 nucleotides.

[0216] To facilitate generation of longer constructs, two or more double- stranded linear DNA molecules and / or gene fragments that are designed with overlapping sequences on the 3 ' strands may be assembled together using methods known in art. For example, the Gibson Assembly™ Method (Synthetic Genomics, Inc., La Jolla, CA) may be performed with the use of a mesophilic exonuclease that cleaves bases from the 5 '-end of the double- stranded DNA fragments, followed by annealing of the newly formed complementary single- DB1 / 154817694.1 90Attorney Docket No.: 116983-5131-WO stranded 3 '-ends, polymerase-dependent extension to fill in any single- stranded gaps, and finally, covalent joining of the DNA segments by a DNA ligase.

[0217] In another aspect, nucleic acid cancer vaccines of the present disclosure comprising, e.g., mRNAs encoding the peptide epitopes, may be made using chemical synthesis of the RNA. Methods, for instance, involve annealing a first polynucleotide comprising an open reading frame encoding the polypeptide and a second polynucleotide comprising a 5'-UTR to a complementary polynucleotide conjugated to a solid support. The 3 '-terminus of the second polynucleotide is then ligated to the 5 '-terminus of the first polynucleotide under suitable conditions. Suitable conditions include the use of a DNA Ligase. The ligation reaction produces a first ligation product. The 5' terminus of a third polynucleotide comprising a 3'- UTR is then ligated to the 3 '-terminus of the first ligation product under suitable conditions. Suitable conditions for the second ligation reaction include an RNA Ligase. A second ligation product is produced in the second ligation reaction. The second ligation product is released from the solid support to produce an mRNA encoding a polypeptide of interest. In some embodiments the mRNA is between 30 and 1000 nucleotides.

[0218] An mRNA encoding one or more peptide epitopes may also be prepared by binding a first nucleic acid comprising an open reading frame encoding the nucleic acid to a second nucleic acid comprising 3'-UTR to a complementary nucleic acid conjugated to a solid support. The 5 '-terminus of the second nucleic acid is ligated to the 3 '-terminus of the first nucleic acid under suitable conditions (including, e.g., a DNA Ligase). The method produces a first ligation product. A third nucleic acid comprising a 5'-UTR is ligated to the first ligation product under suitable conditions (including, e.g., an RNA Ligase, such as T4 RNA) to produce a second ligation product. The second ligation product is released from the solid support to produce an mRNA encoding one or more peptide epitopes.

[0219] In some embodiments the first nucleic acid features a 5 '-triphosphate and a 3'- OH. In other embodiments the second nucleic acid comprises a 3'-OH. In yet other embodiments, the third nucleic acid comprises a 5 '-triphosphate and a 3'-OH. The second nucleic acid may also include a 5 '-cap structure. The method may also involve the further step of ligating a fourth nucleic acid comprising a poly-A region at the 3 '-terminus of the third nucleic acid. The fourth nucleic acid may comprise a 5 '-triphosphate. DB1 / 154817694.1 91Attorney Docket No.: 116983-5131-WO

[0220] The method may or may not comprise reverse phase purification. The method may also include a washing step wherein the solid support is washed to remove unreacted nucleic acids. The solid support may be, for instance, a capture resin. In some embodiments the method involves dT purification.

[0221] In accordance with the present disclosure, template DNA encoding the nucleic acid (e.g., mRNA) cancer vaccines of the present disclosure includes an open reading frame (ORF) encoding one or more peptide epitopes. In some embodiments, the template DNA includes an ORF of up to 1000 nucleotides, e.g., about 10-350, 30-300 nucleotides or about 50-250 nucleotides. In some embodiments, the template DNA includes an ORF of about 150 nucleotides. In some embodiments, the template DNA includes an ORF of about 200 nucleotides.

[0222] In some embodiments, IVT transcripts are purified from the components of the IVT reaction mixture after the reaction takes place. For example, the crude IVT mix may be treated with RNase-free DNase to digest the original template. The nucleic acid (e.g., mRNA) can be purified using methods known in the art, including but not limited to, precipitation using an organic solvent or column based purification method. Commercial kits are available to purify RNA, e.g., MEGACLEAR™ Kit (Ambion, Austin, TX). The nucleic acid (e.g., mRNA) can be quantified using methods known in the art, including but not limited to, commercially available instruments, e.g., NanoDrop. Purified nucleic acids (e.g., mRNAs) can be analyzed, for example, by agarose gel electrophoresis to confirm the nucleic acid is the proper size and / or to confirm that no degradation of the nucleic acid has occurred. G. Untranslated Regions (UTRs)

[0223] Untranslated regions (UTRs) are sections of a nucleic acid before a start codon (5' UTR) and after a stop codon (3' UTR) that are not translated. In some embodiments, a nucleic acid (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprising an open reading frame (ORF) encoding one or more peptide epitopes further comprises one or more UTR (e.g., a 5' UTR or functional fragment thereof, a 3' UTR or functional fragment thereof, or a combination thereof).

[0224] A UTR can be homologous or heterologous to the coding region in a nucleic acid. In some embodiments, the UTR is homologous to the ORF encoding the one or more peptide epitopes. In some embodiments, the UTR is heterologous to the ORF encoding the DB1 / 154817694.1 92Attorney Docket No.: 116983-5131-WO one or more peptide epitopes. In some embodiments, the nucleic acid comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences. In some embodiments, the nucleic acid comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.

[0225] In some embodiments, the 5' UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized. In some embodiments, the 5' UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.

[0226] UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization, and / or translation efficiency. A nucleic acid comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some embodiments, a functional fragment of a 5' UTR or 3' UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.

[0227] Natural 5' UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes.5' UTRs also have been known to form secondary structures that are involved in elongation factor binding.

[0228] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a nucleic acid. For example, introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of nucleic acids in hepatic cell lines or liver. Likewise, use of 5' UTRs from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-l, CD36), for myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CDl lb, MSR, Fr-l, i- NOS), for leukocytes (e.g., CD45, CD 18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin), and for lung epithelial cells (e.g., SP-A / B / C / D).

[0229] In some embodiments, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, an encoded DB1 / 154817694.1 93Attorney Docket No.: 116983-5131-WO polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new nucleic acid.

[0230] In some embodiments, the 5' UTR and the 3' UTR can be heterologous. In some embodiments, the 5' UTR can be derived from a different species than the 3' UTR. In some embodiments, the 3' UTR can be derived from a different species than the 5' UTR.

[0231] International Patent Application No. PCT / US2014 / 021522 (Publ. No. WO / 2014 / 164253) provides a listing of exemplary UTRs that may be utilized in the nucleic acids of the present disclosure as flanking regions to an ORF. This publication is incorporated by reference herein for this purpose.

[0232] Additional exemplary UTRs that may be utilized in the nucleic acids of the present disclosure include, but are not limited to, one or more 5' UTRs and / or 3' UTRs derived from the nucleic acid sequence of: a globin, such as an a- or b-globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-b) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV; e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUTl (human glucose transporter 1)); an actin (e.g., human a or b actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the b subunit of mitochondrial EU-ATP synthase); a growth hormone (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 α1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a b-Fl-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen (e.g., collagen type I, alpha 2 (CollA2), collagen type I, alpha 1 (CollAl), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (C0I6AI)); a ribophorin (e.g., ribophorin I DB1 / 154817694.1 94Attorney Docket No.: 116983-5131-WO (RPNI)); a low density lipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-like cytokine factor (e.g., Nntl); calreticulin (Calr); a procollagen-lysine, 2- oxoglutarate 5- dioxygenase 1 (Plodl); and a nucleobindin (e.g., Nucbl).

[0233] In some embodiments, the 5' UTR is selected from the group consisting of a b- globin 5' UTR; a 5' UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5' UTR; a hydroxysteroid (17-b) dehydrogenase (HSD17B4) 5' UTR; a Tobacco etch virus (TEV) 5' UTR; a Venezuelan equine encephalitis virus (TEEV) 5' UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5' UTR; a heat shock protein 70 (Hsp70) 5' UTR; a eIF4G 5' UTR; a GLUT15' UTR; functional fragments thereof and any combination thereof.

[0234] In some embodiments, the 3' UTR is selected from the group consisting of a b- globin 3' UTR; a CYBA 3' UTR; an albumin 3' UTR; a growth hormone (GH) 3' UTR; a VEEV 3' UTR; a hepatitis B virus (HBV) 3' UTR; a-globin 3' UTR; a DEN 3' UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3' UTR; an elongation factor 1 al (EEF1A1) 3' ETTR; a manganese superoxide dismutase (MnSOD) 3' ETTR; a b subunit of mitochondrial H(+)-ATP synthase (b- mRNA) 3' UTR; a GLUT13' UTR; a MEF2A 3' UTR; a b-Fl-ATPase 3' UTR; functional fragments thereof and combinations thereof.

[0235] Wild-type UTRs derived from any gene or mRNA can be incorporated into the nucleic acids of the disclosure. In some embodiments, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some embodiments, variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.

[0236] Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc.20138(3):568-82, and sequences available at www.addgene.org / Derrick_Rossi / , the contents of each are incorporated herein by reference in their entirety. UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and / or 3' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3' UTRs. DB1 / 154817694.1 95Attorney Docket No.: 116983-5131-WO

[0237] In some embodiments, the nucleic acid may comprise multiple UTRs, e.g., a double, a triple or a quadruple 5' UTR or 3' UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double beta- globin 3' UTR can be used (see, for example, US2010 / 0129877, the contents of which are incorporated herein by reference for this purpose).

[0238] The nucleic acids of the disclosure can comprise combinations of features. For example, the ORF can be flanked by a 5' UTR that comprises a strong Kozak translational initiation signal and / or a 3' UTR comprising an oligo(dT) sequence for templated addition of a poly- A tail. A 5' UTR can comprise a first nucleic acid fragment and a second nucleic acid fragment from the same and / or different UTRs (see, e.g., US2010 / 0293625, herein incorporated by reference in its entirety for this purpose).

[0239] Other non-UTR sequences can be used as regions or subregions within the nucleic acids of the disclosure. For example, introns or portions of intron sequences can be incorporated into the nucleic acids of the disclosure. Incorporation of intronic sequences can increase protein production as well as nucleic acid expression levels. In some embodiments, the nucleic acid of the disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun.2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the nucleic acid comprises an IRES instead of a 5' UTR sequence. In some embodiments, the nucleic acid comprises an ORF and a viral capsid sequence. In some embodiments, the nucleic acid comprises a synthetic 5' UTR in combination with a non- synthetic 3' UTR.

[0240] In some embodiments, the UTR can also include at least one translation enhancer nucleic acid, translation enhancer element, or translational enhancer elements (collectively, “TEE,” which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can include those described in US2009 / 0226470, incorporated herein by reference in its entirety for this purpose, and others known in the art. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some embodiments, the 5' UTR comprises a TEE. In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation. In one non-limiting example, the TEE comprises the TEE DB1 / 154817694.1 96Attorney Docket No.: 116983-5131-WO sequence in the 5 '-leader of the Gtx homeodomain protein. See Chappell et al., PNAS 2004 101:9590-9594, incorporated herein by reference in its entirety for this purpose.

[0241] The terms “translational enhancer polynucleotide” or “translation enhancer polynucleotide sequence” refer to a nucleic acid that includes one or more of the TEE provided herein and / or known in the art (see, e.g., US6310197, US6849405, US7456273, US7183395, US2009 / 0226470, US2007 / 0048776, US2011 / 0124100, US2009 / 0093049, US2013 / 0177581, W 02009 / 075886, W02007 / 025008, W02012 / 009644, WO2001 / 055371, WO1999 / 024595, EP2610341A1, and EP2610340A1; the contents of each of which are incorporated herein by reference in their entirety for this purpose), or their variants, homologs, or functional derivatives. In some embodiments, the nucleic acid of the disclosure comprises one or multiple copies of a TEE. The TEE in a translational enhancer nucleic acid can be organized in one or more sequence segments. A sequence segment can harbor one or more of the TEEs provided herein, with each TEE being present in one or more copies. When multiple sequence segments are present in a translational enhancer nucleic acid, they can be homogenous or heterogeneous. Thus, the multiple sequence segments in a translational enhancer nucleic acid can harbor identical or different types of the TEE provided herein, identical or different number of copies of each of the TEE, and / or identical or different organization of the TEE within each sequence segment. In one embodiment, the nucleic acid of the disclosure comprises a translational enhancer nucleic acid sequence.

[0242] In some embodiments, a 5' UTR and / or 3' UTR comprising at least one TEE described herein can be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector. In some embodiments, a 5' UTR and / or 3' UTR of a polynucleotide of the disclosure comprises a TEE or portion thereof described herein. In some embodiments, the TEEs in the 3' UTR can be the same and / or different from the TEE located in the 5' UTR.

[0243] In some embodiments, a 5' UTR and / or 3' UTR of a nucleic acid of the disclosure can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In one embodiment, the 5' UTR of a nucleic acid of the DB1 / 154817694.1 97Attorney Docket No.: 116983-5131-WO disclosure can include 1-60, 1-55, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. The TEE sequences in the 5' UTR of the nucleic acid of the disclosure can be the same or different TEE sequences. A combination of different TEE sequences in the 5' UTR of the nucleic acid of the disclosure can include combinations in which more than one copy of any of the different TEE sequences are incorporated.

[0244] In some embodiments, the 5' UTR and / or 3' UTR comprises a spacer to separate two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and / or other spacers known in the art (e.g., in multiples of three nucleotides). As another non-limiting example, the 5' UTR and / or 3' UTR comprises a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more than 10 times in the 5' UTR and / or 3' UTR, respectively. In some embodiments, the 5' UTR and / or 3' UTR comprises a TEE sequence-spacer module repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. H. 3 ' UTR and the AU Rich Elements

[0245] In certain embodiments, a nucleic acid of the present disclosure (e.g., a nucleic acid encoding a peptide epitope of the disclosure) further comprises a 3' UTR.

[0246] A 3'-UTR is the section of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3'-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. In one embodiment, the 3'- UTR useful for the disclosure comprises a binding site for regulatory proteins or microRNAs. In some embodiments, the 3'-UTR has a silencer region, which binds to repressor proteins and inhibits the expression of the mRNA. In other embodiments, the 3'-UTR comprises an AU-rich element (AREs). Proteins bind AREs to affect the stability or decay rate of transcripts in a localized manner or affect translation initiation. In other embodiments, the 3'- UTR comprises the sequence AAUAAA that directs addition of several hundred adenine residues called the poly-A tail to the end of the mRNA transcript.

[0247] Natural or wild type 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes DB1 / 154817694.1 98Attorney Docket No.: 116983-5131-WO with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM- CSF and TNF-a. Class III ARES do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0248] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of nucleic acids of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection. I. Regions having a 5' Cap

[0249] The nucleic acid cancer vaccine described herein may be an mRNA cancer vaccine comprising one or more mRNA having open reading frames that encode peptide epitopes. Each of these mRNA may have a 5' Cap.

[0250] The 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns during mRNA splicing. DB1 / 154817694.1 99Attorney Docket No.: 116983-5131-WO

[0251] Endogenous mRNA molecules can be 5 '-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule (cap). This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue (cap-0). The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-0- methylated (e.g., with a 2'-hydroxy group on the first ribose sugar (cap-l); or with a 2'- hydroxy group on the first two ribose sugars (cap-2)).5 '-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0252] In some embodiments, nucleic acids of the present disclosure (e.g., a nucleic acid encoding a peptide epitope) incorporate a cap moiety.

[0253] In some embodiments, nucleic acids of the present disclosure (e.g., a nucleic acid encoding a peptide epitope) comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with a-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides can be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0254] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and / or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'- caps in their chemical structure, while retaining cap function. Cap analogs can be chemically {i.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the disclosure.

[0255] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an N7 methyl DB1 / 154817694.1 100Attorney Docket No.: 116983-5131-WO group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'- guanosine (m7G-3'mppp-G); which can equivalently be designated 3' O-Me- m7G(5')ppp(5')G). The 3'-O atom of the other, unmodified, guanine becomes linked to the 5'- terminal nucleotide of the capped polynucleotide. The N7- and 3'-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.

[0256] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0- methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm- ppp-G).

[0257] In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety for this purpose.

[0258] In another embodiment, the cap is a cap analog is a N7-(4- chlorophenoxyethyl) substituted dicucleotide form of a cap analog known in the art and / or described herein. Non limiting examples of a N7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4- chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by reference in its entirety for this purpose). In another embodiment, a cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0259] While cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '- cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, can lead to reduced translational competency and reduced cellular stability.

[0260] Nucleic acids of the disclosure (e.g., a nucleic acids encoding peptide antigens) can also be capped post-manufacture (whether through IVT or chemical synthesis), using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally DB1 / 154817694.1 101Attorney Docket No.: 116983-5131-WO or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 'cap structures are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5 'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O- methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5'- terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O- methyl. Such a structure is termed the cap-l structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro- inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap-0), 7mG(5')ppp(5')NlmpNp (cap-l), and 7mG(5')-ppp(5')NlmpN2mp (cap-2).

[0261] As a non-limiting example, capping chimeric nucleic acids post-manufacture can be more efficient as nearly 100% of the chimeric nucleic acids can be capped. This is in contrast to -80% when a cap analog is linked to a chimeric nucleic acids in the course of an in vitro transcription reaction.

[0262] According to the present disclosure, 5' terminal caps can include endogenous caps or cap analogs. According to the present disclosure, a 5' terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl- guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. J. Poly-A Tails

[0263] In some embodiments, the nucleic acids of the present disclosure (e.g., a nucleic acid encoding peptide epitopes) further comprise a poly-A tail. In further embodiments, terminal groups on the poly-A tail can be incorporated for stabilization. In other embodiments, a poly- A tail comprises des-3' hydroxyl tails. DB1 / 154817694.1 102Attorney Docket No.: 116983-5131-WO

[0264] During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a nucleic acid such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues long. In some embodiments, the poly-A tail comprises about 100 nucleotides.

[0265] Poly-A tails can also be added after the construct is exported from the nucleus.

[0266] According to the present disclosure, terminal groups on the poly-A tail can be incorporated for stabilization· Polynucleotides of the present disclosure can include des-3' hydroxyl tails. They can also include structural moieties or 2'-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol.15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in its entirety for this purpose).

[0267] The nucleic acids of the present disclosure can be designed to encode transcripts with alternative poly-A tail structures including histone mRNA. According to Norbury, “[t]erminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication. These mRNAs are distinguished by their lack of a 3 ' poly-A tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs” (Norbury,“Cytoplasmic RNA: a case of the tail wagging the dog,” Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi:l0.l038 / nrm3645) the contents of which are incorporated herein by reference in its entirety for this purpose.

[0268] Unique poly-A tail lengths provide certain advantages to the nucleic acids of the present disclosure. Generally, the length of a poly-A tail, when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length ( e.g at least or greater than about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides). In DB1 / 154817694.1 103Attorney Docket No.: 116983-5131-WO some embodiments, the nucleic acid or region thereof includes from about 15 to about 3,000 nucleotides ( e.g from 15 to 50, 15 to 100, 15 to 200, 15 to 300, 15 to 400, 15 to 500, 15 to 600, 15 to 700, 15 to 800, 15 to 900, 15 to 1000, 15 to 1200, 15 to 1400, 15 to 1500, 15 to 1800, 15 to 2000, 15 to 2500, 15 to 3000, 50 to 100, 50 to 200, 50 to 300, 50 to 400, 50 to 500, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 50 to 1200, 50 to 1400, 50 to 1500, 50 to 1800, 50 to 2000, 50 to 2500, 50 to 3000, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 100 to 600, 100 to 700, 100 to 800, 100 to 900, 100 to 1000, 100 to 1200, 100 to 1400, 100 to 1500, 100 to 1800, 100 to 2000, 100 to 2500, 100 to 3000, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200, to 800, 200 to 900, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 2500, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 2500, 1000 to 3000, 1500 to 3000, 2500 to 3000, or 2000 to 3000 nucleotides).

[0269] In some embodiments, the poly-A tail is designed relative to the length of the overall nucleic acid or the length of a particular region of the nucleic acid. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the nucleic acids.

[0270] In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the nucleic acid or feature thereof. The poly-A tail can also be designed as a fraction of the nucleic acid to which it belongs. In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of nucleic acids for Poly-A binding protein can enhance expression.

[0271] Additionally, multiple distinct nucleic acids can be linked together via the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '- terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at l2hr, 24hr, 48hr, 72hr, and / or day 7 post transfection.

[0272] In some embodiments, the nucleic acids of the present disclosure are designed to include a poly-A-G Quartet region. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant DB1 / 154817694.1 104Attorney Docket No.: 116983-5131-WO nucleic acid is assayed for stability, protein production, and other parameters including half- life at various time points. It has been discovered that the poly-A-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone. K. Start codon region

[0273] The disclosure also includes a nucleic acid that comprises both a start codon region and the nucleic acid described herein (e.g., a nucleic acid comprising a nucleotide sequence encoding peptide epitopes). In some embodiments, the nucleic acids of the present disclosure can have regions that are analogous to or function like a start codon region.

[0274] In some embodiments, the translation of a nucleic acid can initiate on a codon that is not the start codon AUG. Translation of the nucleic acid can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 20105:11; the contents of each of which are herein incorporated by reference in its entirety for this purpose).

[0275] As a non-limiting example, the translation of a nucleic acid begins on the alternative start codon ACG. As another non-limiting example, nucleic acid translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a nucleic acid begins on the alternative start codon GTG or GUG.

[0276] Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and / or the structure of the nucleic acid. (See, e.g., Matsuda and Mauro PLoS ONE, 20105:11; the contents of which are herein incorporated by reference in its entirety for this purpose). Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length, and / or structure of a polynucleotide.

[0277] In some embodiments, a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) nucleic acids and exon-junction complexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agents DB1 / 154817694.1 105Attorney Docket No.: 116983-5131-WO LNA polynucleotides and EJCs (PLoS ONE, 20105:11); the contents of which are herein incorporated by reference in its entirety for this purpose).

[0278] In another embodiment, a masking agent can be used to mask a start codon of a nucleic acid in order to increase the likelihood that translation will initiate on an alternative start codon. In some embodiments, a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

[0279] In another embodiment, the start codon of a nucleic acid can be removed from the nucleic acid sequence in order to have the translation of the nucleic acid begin on a codon that is not the start codon. Translation of the nucleic acid can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. In a non-limiting example, the start codon ATG or AUG is removed as the first 3 nucleotides of the nucleic acid sequence in order to have translation initiate on a downstream start codon or alternative start codon. The nucleic acid sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and / or alternative start codons in order to control or attempt to control the initiation of translation, the length of the nucleic acid and / or the structure of the nucleic acid. L. Stop Codon Region

[0280] The disclosure also includes a nucleic acid that comprises both a stop codon region and the nucleic acid described herein (e.g., a nucleic acid encoding peptide epitopes). In some embodiments, the nucleic acids of the present disclosure can include at least two stop codons before the 3' untranslated region (UTR). The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In some embodiments, the nucleic acids of the present disclosure include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon. In a further embodiment the addition stop codon can be TAA or UAA. In another embodiment, the nucleic acids of the present disclosure include three consecutive stop codons, four stop codons, or more. DB1 / 154817694.1 106Attorney Docket No.: 116983-5131-WO M. Insertions and Substitutions

[0281] The disclosure also includes a nucleic acid of the present disclosure that further comprises insertions and / or substitutions.

[0282] In some embodiments, the 5' UTR of the nucleic acid can be replaced by the insertion of at least one region and / or string of nucleosides of the same base. The region and / or string of nucleotides can include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides and the nucleotides can be natural and / or unnatural. As a non-limiting example, the group of nucleotides can include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and / or combinations thereof.

[0283] In some embodiments, the 5' UTR of the nucleic acid can be replaced by the insertion of at least two regions and / or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein, and / or combinations thereof. For example, the 5' UTR can be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5' UTR can be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.

[0284] In some embodiments, the nucleic acid can include at least one substitution and / or insertion downstream of the transcription start site that can be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and / or insertion can occur downstream of the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site can affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its entirety for this purpose). The modification, substitution, and / or insertion of at least one nucleoside can cause a silent mutation of the sequence or can cause a mutation in the amino acid sequence.

[0285] In some embodiments, the nucleic acid can include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least DB1 / 154817694.1 107Attorney Docket No.: 116983-5131-WO 10, at least 11, at least 12, or at least 13 guanine bases downstream of the transcription start site.

[0286] In some embodiments, the nucleic acid can include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 guanine bases in the region just downstream of the transcription start site. As a non-limiting example, if the nucleotides in the region are GGGAGA, the guanine bases can be substituted by at least 1, at least 2, at least 3, or at least 4 adenine nucleotides. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases can be substituted by at least 1, at least 2, at least 3, or at least 4 cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases can be substituted by at least 1, at least 2, at least 3, or at least 4 thymine, and / or any of the nucleotides described herein.

[0287] In some embodiments, the nucleic acid can include at least one substitution and / or insertion upstream of the start codon. For the purpose of clarity, one of skill in the art would appreciate that the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins. The nucleic acid can include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 substitutions and / or insertions of nucleotide bases. The nucleotide bases can be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4, or at least 5 locations upstream of the start codon. The nucleotides inserted and / or substituted can be the same base (e.g., all A, or all C, or all T, or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T, or A, C and T) or at least four different bases.

[0288] As a non-limiting example, the guanine base upstream of the coding region in the nucleic acid can be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, the substitution of guanine bases in the nucleic acid can be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344): 499-503; the contents of which is herein incorporated by reference in its entirety for this purpose). As a non-limiting example, at least 5 nucleotides can be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides can be the same base type.

[0289] According to the present disclosure, two regions or parts of a chimeric nucleic acid may be joined or ligated, for example, using triphosphate chemistry. In some DB1 / 154817694.1 108Attorney Docket No.: 116983-5131-WO embodiments, a first region or part of 100 nucleotides or less is chemically synthesized with a 5'- monophosphate and terminal 3'-desOH or blocked OH. If the region is longer than 80 nucleotides, it may be synthesized as two or more strands that will subsequently be chemically linked by ligation. If the first region or part is synthesized as a non-positionally modified region or part using IVT, conversion to the 5 '-monophosphate with subsequent capping of the 3 '-terminus may follow. Monophosphate protecting groups may be selected from any of those known in the art. A second region or part of the chimeric nucleic acid may be synthesized using either chemical synthesis or IVT methods, e.g., as described herein. IVT methods may include use of an RNA polymerase that can utilize a primer with a modified cap. Alternatively, a cap may be chemically synthesized and coupled to the IVT region or part.

[0290] It is noted that for ligation methods, ligation with DNA T4 ligase followed by DNAse treatment (to eliminate the DNA splint required for DNA T4 Ligase activity) should readily prevent the undesirable formation of concatenation products. The entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then it is preferable that such region or part comprise a phosphate-sugar backbone. Ligation may be performed using any appropriate technique, such as enzymatic ligation, click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art. In some embodiments, the ligation is directed by a complementary oligonucleotide splint. In some embodiments, the ligation is performed without a complementary oligonucleotide splint. III. TIL Manufacturing Processes

[0016] An exemplary family of TIL processes known as Gen 2 (also known as process 2A) containing some of these features is depicted in Figures 1 and 2. An embodiment of Gen 2 is shown in Figure 2.

[0017] As discussed herein, the present invention can include a step relating to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health. As generally outlined herein, TILs are generally taken from a patient sample and manipulated to DB1 / 154817694.1 109Attorney Docket No.: 116983-5131-WO expand their number prior to transplant into a patient. In some embodiments, the TILs may be optionally genetically manipulated as discussed below.

[0018] In some embodiments, the TILs may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.

[0019] In some embodiments, the first expansion (including processes referred to as the pre-REP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shorted to 7 to 14 days, as discussed in detail below as well as in the examples and figures. In some embodiments, the first expansion (for example, an expansion described as Step B in Figure 1) is shortened to 11 days and the second expansion (for example, an expansion as described in Step D in Figure 1) is shortened to 11 days. In some embodiments, the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.

[0020] The “Step” Designations A, B, C, etc., below are in reference to Figure 1 and in reference to certain embodiments described herein. The ordering of the Steps below and in Figure 1 is exemplary and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps is contemplated by the present application and the methods disclosed herein. A. STEP A: Obtain Patient Tumor Sample

[0021] In general, TILs are initially obtained from a patient tumor sample and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.

[0022] A patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In some embodiments, multilesional sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells includes multilesional sampling (i.e., obtaining samples from one or more tumor sites and / or locations in the patient, as well as one or more tumors in the same location or in close DB1 / 154817694.1 110Attorney Docket No.: 116983-5131-WO proximity). In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung carcinoma (NSCLC). The solid tumor may be of skin tissue. In some embodiments, useful TILs are obtained from a melanoma.

[0023] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3being particularly useful. In some embodiments, the TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicine, 30 units / mL of DNase and 1.0 mg / mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No.2012 / 0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.

[0024] Tumor dissociating enzyme mixtures can include one or more dissociating (digesting) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.

[0025] In some embodiments, the dissociating enzymes are reconstituted from lyophilized enzymes. In some embodiments, lyophilized enzymes are reconstituted in an amount of sterile buffer such as HBSS. DB1 / 154817694.1 111Attorney Docket No.: 116983-5131-WO

[0026] In some instances, collagenase (such as animal free- type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiment, after reconstitution the collagenase stock ranges from about 100 PZ U / mL-about 400 PZ U / mL, e.g., about 100 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL-about 350 PZ U / mL, about 100 PZ U / mL-about 300 PZ U / mL, about 150 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL, about 150 PZ U / mL, about 200 PZ U / mL, about 210 PZ U / mL, about 220 PZ U / mL, about 230 PZ U / mL, about 240 PZ U / mL, about 250 PZ U / mL, about 260 PZ U / mL, about 270 PZ U / mL, about 280 PZ U / mL, about 289.2 PZ U / mL, about 300 PZ U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0027] In some embodiments, neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, after reconstitution the neutral protease stock ranges from about 100 DMC / mL-about 400 DMC / mL, e.g., about 100 DMC / mL-about 400 DMC / mL, about 100 DMC / mL-about 350 DMC / mL, about 100 DMC / mL-about 300 DMC / mL, about 150 DMC / mL-about 400 DMC / mL, about 100 DMC / mL, about 110 DMC / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0028] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, after reconstitution the DNase I stock ranges from about 1 KU / mL-10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0029] In some embodiments, the stock of enzymes is variable and the concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digest cocktail is adjusted based on the determined stock concentration. DB1 / 154817694.1 112Attorney Docket No.: 116983-5131-WO

[0030] In some embodiment, the enzyme mixture includes about 10.2-ul of neutral protease (0.36 DMC U / mL), 21.3 µL of collagenase (1.2 PZ / mL) and 250-ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0031] As indicated above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumors are not fragmented. In some embodiments, the solid tumors are not fragmented and are subjected to enzymatic digestion as whole tumors. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2 with rotation. In some embodiments, the tumors are digested overnight with constant rotation. In some embodiments, the tumors are digested overnight at 37°C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.

[0032] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0033] In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for the collagenase is a 100 mg / mL 10X working stock.

[0034] In some embodiments, the enzyme mixture comprises DNAse. In some embodiments, the working stock for the DNAse is a 10,000 IU / mL 10X working stock.

[0035] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock for the hyaluronidase is a 10 mg / mL 10X working stock.

[0036] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0037] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase. DB1 / 154817694.1 113Attorney Docket No.: 116983-5131-WO

[0038] In general, the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population.

[0039] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0040] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1). In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm3. In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm3to about 1500 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

[0041] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some DB1 / 154817694.1 114Attorney Docket No.: 116983-5131-WO embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the tumors are 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumors are 1 mm × 1 mm × 1 mm. In some embodiments, the tumors are 2 mm × 2 mm × 2 mm. In some embodiments, the tumors are 3 mm × 3 mm × 3 mm. In some embodiments, the tumors are 4 mm × 4 mm × 4 mm.

[0042] In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and / or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.

[0043] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO2and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[0044] In some embodiments, the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population. DB1 / 154817694.1 115Attorney Docket No.: 116983-5131-WO

[0045] In some emb...

Claims

Attorney Docket No.: 116983-5131-WO WHAT IS CLAIMED IS: CLAIMS 1. A method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient and dividing the tumor sample into a first portion and a second portion; b) expanding a population of TILs from the first portion of the tumor sample into a therapeutic population of TILs; c) making a cancer vaccine using the second portion of the tumor sample; d) administering the therapeutic population of TILs to the patient; and e) administering the cancer vaccine to the patient.

2. The method of claim 1, wherein making a cancer vaccine comprises: i) identifying between 1-1,000 personalized cancer neoantigens by analyzing the second portion of the tumor sample; and ii) preparing a cancer vaccine wherein the cancer vaccine comprises portions of the 1- 1,000 personalized cancer neoantigens.

3. The method of claim 2, wherein analyzing the second portion of the tumor sample comprises obtaining the transcriptome of a tumor cell in the second portion of the tumor sample.

4. The method of claim 3, wherein analyzing the second portion of the tumor sample further comprises comparing the transcriptome of the tumor cell to the transcriptome of a reference cell.

5. The method of claim 4, wherein the reference cell is a non-tumor cell in the second portion of the tumor sample.

6. The method of any one of claims 3-5, wherein obtaining the transcriptome of the tumor cell comprises single-cell sequencing.

7. The method of any one of claims 1-6, wherein the cancer vaccine comprises portions of 1-200 personalized cancer neoantigens.

8. The method of any one of claims 1-6, wherein the cancer vaccine comprises portions of 1-100 personalized cancer neoantigens. DB1 / 154817694.1 250Attorney Docket No.: 116983-5131-WO 9. The method of any one of claims 1-8, wherein the cancer vaccine is a nucleic acid cancer vaccine comprising one or more nucleic acids each having one or more open reading frames.

10. The method of any one of claims 1-9, wherein the cancer vaccine comprises one or more nucleic acids each having one or more open reading frames encoding 1-1,000 peptide epitopes, wherein each of the peptide epitopes is a portion of one of the 1- 1,000 personalized cancer neoantigens from the patient.

11. The method of claim 10, wherein the minimum length of any peptide epitope is 8-13 amino acids.

12. The method of claim 10 or 11, wherein the maximum length of any peptide epitope is 31-35 amino acids.

13. The method of any one of claims 10-12, wherein each of the peptide epitopes is encoded by a separate open reading frame.

14. The method of any one of claims 10-13, wherein the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 1-1,000 peptide epitopes.

15. The method of any one of claims 1-14, wherein the cancer vaccine is a DNA cancer vaccine.

16. The method of any one of claims 1-14, wherein the cancer vaccine is an RNA cancer vaccine.

17. The method of claim 16, wherein the cancer vaccine is an mRNA cancer vaccine, and wherein the one or more nucleic acids are mRNA.

18. The method of claim 17, wherein the one or more mRNA each comprise a 5' UTR and / or a 3' UTR.

19. The method of claim 17 or 18, wherein the one or more mRNA each comprise a poly- A tail.

20. The method of claim 19, wherein the poly-A tail comprises about 100 nucleotides.

21. The method of any one of claims 17-20, wherein the one or more mRNA each comprise a cap structure or a modified cap structure.

22. The method of claim 21, wherein the cap structure or the modified cap structure is a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, or a 5' cap-2 structure.

23. The method of any one of claims 17-22, wherein the one or more mRNA comprise at least one chemical modification. DB1 / 154817694.1 251Attorney Docket No.: 116983-5131-WO 24. The method of claim 23, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2- thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 5-methylpseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, and 2'-O-methyl uridine.

25. The method of claim 23 or claim 24, wherein the one or more mRNA is fully modified.

26. The method of any one of claims 10-25, wherein the one or more nucleic acids encode 1-5 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20- 30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60- 70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-200 peptide epitopes, 200-300 peptide epitopes, 300-400 peptide epitopes, 400-500 peptide epitopes, 500-600 peptide epitopes, 600-700 peptide epitopes, 700-800 peptide epitopes, 800-900 peptide epitopes, or 900-1,000 peptide epitopes.

27. The method of any one of claims 10-26, wherein each of the peptide epitopes is encoded by a separate open reading frame.

28. The method of any one of claims 10-27, wherein the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 5-130 peptide epitopes.

29. The method of any one of claims 10-28, wherein one or more of the following conditions are met: a) the 1-1,000 peptide epitopes are interspersed by cleavage sensitive sites; and / or b) each peptide epitope is linked directly to one another without a linker; and / or c) each peptide epitope is linked to one another with a single amino acid linker; and / or d) each peptide epitope is linked to one another with a short peptide linker; and / or e) each peptide epitope comprises 8-35 amino acids and includes one or more SNP mutations; and / or f) each peptide epitope comprises 8-35 amino acids and includes a mutation causing a unique expressed peptide sequence; and / or DB1 / 154817694.1 252Attorney Docket No.: 116983-5131-WO b) none of the peptide epitopes have a highest affinity for class II MHC molecules from a subject; and / or c) the nucleic acid encoding the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes; and / or d) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; and / or e) no class II MHC molecule peptide epitopes are present; and / or f) at least 30% of the peptide epitopes have a highest affinity for class I MHC molecules and / or class II MHC class molecules from a subject; and / or g) at least 50% of the peptide epitopes have a probability percent rank greater than 0.5% for HLA-A, HLA-B, and / or DRB1.

30. The method of any one of claims 10-29, wherein at least one of the peptide epitopes is a predicted T cell reactive epitope.

31. The method of any one of claims 10-30, wherein at least one of the peptide epitopes is a predicted B cell reactive epitope.

32. The method of any one of claims 10-31, wherein the peptide epitopes comprise a combination of predicted T cell reactive epitopes and predicted B cell reactive epitopes.

33. The method of any one of claims 10-32, wherein the peptide epitopes are predicted T cell reactive epitopes and / or predicted B cell reactive epitopes.

34. The method of any one of claims 10-33, wherein at least one of the peptide epitopes is a predicted neoepitope.

35. The method of any one of claims 10-34, wherein at least one nucleic acid has an open reading frame encoding at least a fragment of one or more traditional cancer antigens or one or more cancer / testis antigens.

36. The method of any one of claims 10-35, wherein each nucleic acid is formulated in a lipid nanoparticle.

37. The method of claim 36, wherein each nucleic acid is formulated in a different lipid nanoparticle.

38. The method of claim 36, wherein each nucleic acid is formulated in the same lipid nanoparticle. DB1 / 154817694.1 253Attorney Docket No.: 116983-5131-WO 39. The method of any one of claims 10-38, wherein the total length of the one or more nucleic acids encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, 900- 1,000 amino acids, 1,000-2,000 amino acids, 2,000-3,000 amino acids, 3,000-4,000 amino acids, 4,000-5,000 amino acids, 5,000-6,000 amino acids, 6,000-7,000 amino acids, 7,000-8,000 amino acids, 8,000-9,000 amino acids, or 9,000-10,000 amino acids.

40. The method of any one of claims 1-8, wherein the cancer vaccine is a peptide cancer vaccine.

41. The method of any one of claims 1-40, wherein the cancer vaccine is administered at a dosage level sufficient to deliver between 0.02-1.0 mg of the cancer vaccine to the subject.

42. The method of any one of claims 1-41, wherein the cancer vaccine is administered to the patient after the administration of the therapeutic population of TILs.

43. The method of claim 42, wherein the cancer vaccine is administered to the patient one week to twelve weeks after the administration of the therapeutic population of TILs.

44. The method of claim 42, wherein the cancer vaccine is administered to the patient two weeks after the administration of the therapeutic population of TILs.

45. The method of any one of claims 1-44, wherein the cancer vaccine is administered to the patient once, twice, three times, four times, five times, six times, seven times, eight times, ten times, twelve times, or more.

46. The method of any one of claims 1-45, wherein the cancer vaccine is administered once weekly (QW), once every 2 weeks (Q2W), every 3 weeks (Q3W), or every 4 weeks (Q4W).

47. The method of any one of claims 1-46, wherein the cancer vaccine is administered Q3W for nine times.

48. The method of any one of claims 1-47, wherein the cancer vaccine is administered by intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous administration.

49. The method of claim 48, wherein the cancer vaccine is administered by intramuscular administration. DB1 / 154817694.1 254Attorney Docket No.: 116983-5131-WO 50. The method of any one of claims 1-49, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, metastatic melanoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), a solid malignancy that is micro satellite high (MSI H) / mismatch repair (MMR) deficient, renal cancer, gastric cancer, and tumor mutational burden high tumors.

51. The method of any one of claims 1-49, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), metastatic melanoma, and head and neck squamous cell carcinoma (HNSCC).

52. The use according any one of claims 1-51 for the manufacture of a medicament for use in the treatment of cancer DB1 / 154817694.1 255

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